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Pathophysiology of rhinitis. Lactoferrin and lysozyme in nasal secretions
Abstract
The antimicrobial proteins lactoferrin (Lf) and lysozyme (Ly) are invariably found in nasal secretions. To investigate the cellular sources and the secretory control of these nasal proteins in vivo, 34 adult subjects underwent nasal provocation tests with methacholine (MC), histamine (H), and gustatory stimuli. Nasal lavages were collected and analyzed for total protein (TP), albumin (Alb), Lf, and Ly. MC (25 mg), H (1 mg), and gustatory stimuli (spicy foods) all increased the concentrations of TP, Alb, Lf, and Ly. However, when each protein was assessed as a percentage of TP (i.e., Alb% = Alb/TP; Lf% = Lf/TP; Ly% = Ly/TP), MC and gustatory stimuli, which both induce glandular secretion, selectively augmented Lf% and Ly% without changing Alb%, while H, which primarily increases vascular permeability, increased Alb% without significantly affecting Lf% or Ly%. Gel electrophoresis and immunoblotting analysis of nasal secretions demonstrated both Lf and Ly in cholinergically induced secretions. Furthermore, histochemical analyses of nasal turbinate tissue revealed Lf and Ly colocalization within the serous cells of submucosal glands, providing evidence that both proteins are strictly glandular products within the nasal mucosa. Therefore, both Lf and Ly are produced and secreted from the glands, and their secretion may be pharmacologically regulated in attempts to improve host defenses.
... Type II and immune subsets. Mouse lung biopsies were collected at the indicated time points: naïve, 3,5,7,9,11,14,17,21,60,120, and 240 days post-infection. A sample size of 5 C57/Bl6 WT mice was used for each timepoint. ...
... For these experiments, a sample size of 3 Sftpc-CreER/ROSA-mTmG was pooled together prior to cell sorting. Cells were collected at the same indicated time points (naïve, 3,5,7,9,11,14,17,21,60,120, and 240 days post-infection) and a minimum of 50,000 cells were collected per timepoint. In experiments where isolation of conducting airway epithelium was not possible using Sftpc-CreER/ROSA-mTmG (Fig. 6H) an alternative gating strategy was used to deplete Type II cells. ...
... Single-cell datasets were subsetted based on 'cell type' annotation and changes in gene expression were assessed at different time points after PR8 exposure using the hallmark gene sets from the molecular signature database 57,58 . Cell type annotation was performed using the following cell type-specific markers: Ltf (Serous cells 59,60 ), Krt5, Trp63 (Basal cells 61,62 ), Cldn10, Scgb1a1 (Club cells 63,64 ), Ager (Alveolar Type I cells 65 ), Sftpc, Chil1, Hc, Scid1 (Alveolar Type II cells 66 ), Foxj1 (Ciliated cells 67 ). The R package 'Velocyto' and 'scVelo' 31,68 was used to perform the single-cell trajectory analysis. ...
Epithelial plasticity has been suggested in lungs of mice following genetic depletion of stem cells but is of unknown physiological relevance. Viral infection and chronic lung disease share similar pathological features of stem cell loss in alveoli, basal cell (BC) hyperplasia in small airways, and innate immune activation, that contribute to epithelial remodeling and loss of lung function. We show that a subset of distal airway secretory cells, intralobar serous (IS) cells, are activated to assume BC fates following influenza virus infection. Injury-induced hyperplastic BC (hBC) differ from pre-existing BC by high expression of IL-22Ra1 and undergo IL-22-dependent expansion for colonization of injured alveoli. Resolution of virus-elicited inflammation results in BC to IS re-differentiation in repopulated alveoli, and increased local expression of protective antimicrobial factors, but fails to restore normal alveolar epithelium responsible for gas exchange.
... Well described and abundant proteins in nasal mucus are mainly antimicrobial proteins like lysozyme and lactotransferrin as well as secretory immunoglobulins IgA, IgE, IgG, and albumin. Other proteins include kallikrein, antiproteases, ß-glucuronidase, and α-galactosidase as described in early, mainly ELISA-based, studies [4,5]. Mucus proteins stem from epithelial cells, goblet cells of sero-mucous glands in the submucosal tissue, either by secretion or cell death. ...
... Cellular debris in the mucus can be detected by its DNA or nucleic products such as histones [3]. Another important contributor to the protein composition of the mucus is proteins exudated from plasma, which is regarded as a key defense mechanism [3,4,6,7]. Especially in pathological conditions like allergic rhinitis, lysozyme, and lactotransferrin were shown to be up-regulated after provocation with allergen [4]. These early studies already underlined the importance of the nasal mucus proteome in disease, but were restricted to selected distinct proteins or small groups of proteins. ...
... Another important contributor to the protein composition of the mucus is proteins exudated from plasma, which is regarded as a key defense mechanism [3,4,6,7]. Especially in pathological conditions like allergic rhinitis, lysozyme, and lactotransferrin were shown to be up-regulated after provocation with allergen [4]. These early studies already underlined the importance of the nasal mucus proteome in disease, but were restricted to selected distinct proteins or small groups of proteins. ...
Introduction: Nasal mucus is the first line defense barrier against various pathogens including allergens. Proteins in nasal mucus maybe used as biomarkers for diagnosis or future therapeutic strategies. Proteomics opens the possibility to investigate whole human proteomes.
Areas Covered: We aimed to analyze the existing literature on nasal mucus and nasal secretions proteomic approaches especially in allergic rhinitis. A PubMed/Medline search was conducted entering the following keywords and combinations: “nasal mucus”, “nasal lavage fluid,” nasal secretions,” “nasal swabs,” “allergic rhinitis,” ”proteins,” and “proteomics.”
Expert opinion: The majority of studies focus on single proteins or protein groups mainly using ELISA techniques. Four studies met the criteria using mass spectrometry in the analysis of nasal mucus proteomes in rhinologic diseases. In these studies, 7, 35, 267, and 430 proteins were identified, respectively. These four studies are discussed in this review and put in relation to seven other proteomic studies that focus on nasal lavage fluid and nasal secretions obtained by swabs or filter paper. To put it in a nutshell, proteomics facilitates the investigation of the nasal secretome and its role in healthy and diseased state and as potential biomarkers for new diagnostic or therapeutic approaches.
... Since the reduction of nasal mucosa inflammation and edema is of special interest in acute nasopharyngitis (7), the natural compound lysozyme could have synergistic effects when combined with xylometazoline. Lysozyme is an antimicrobial protein present in healthy nasal secretions (8). The mucous blanket contains lysozyme and immunoglobulins and plays an important role in immune response (9). ...
... This is probably because the medical doctors involved in the study were more likely to include only xylometazoline therapy to these subjects. However, this approach should be changed considering that lysozyme is a natural compound found in nasal secretions (8). The spray containing xylometazoline and lysozyme has proven efficacy and safety and has been registered as an over-the-counter (OTC) medicine in Bosnia and Herzegovina for more than four years (18). ...
Background: Acute nasopharyngitis is a common condition usually accompanied by nasal congestion. The aim of this study was to compare efficacy and safety of the spray containing xylometazoline and lysozyme with spray containing only xylometazoline in the treatment of acute nasopharyngitis.Methods: Prospective, comparative, post-marketing study was performed on subjects with acute nasopharyngitisdivided into xylometazoline+lysozyme or xylometazoline nasal spray groups. Data collection was performed at the baseline before and 30 minutes after the therapy application and seven days after baseline.Main findings: Out of 173 included subjects, 59 were in the xylometazoline+lysozyme and 114 in the xylometazoline group. In both groups nasal patency was significantly improved 30 minutes after the therapy application (p<0.001). In the xylometazoline+lysozyme group all subjects had nasal decongestion within 20 minutes and this was significantly shorter (p=0.037) compared to xylometazoline group where 16 subjects (14%) needed 20 to 120 minutes for nasal decongestion. All adverse events were mild and there was no significant difference in the number of adverse events between the groups.Principal conclusions: Nasal sprays containing xylometazoline with or without lysozyme were effective and safe in the treatment of acute nasopharyngitis. Nasal spray containing xylometazoline with lysozyme showed a faster effect with significantly shorter time to nose decongestion. All recorded adverse events were mild and there was no difference between the groups in the number of recorded adverse events. Key words: nasopharyngitis, nasal obstruction, lysozyme, xylometazoline,nasal sprays
... However, we have recently demonstrated in vitro that fungi isolated from the mucin of CRS patients were not resistant to lysozyme (9) and hypothesised that a deficiency in lysozyme mRNA expression and/or protein secretion may be present in CRS patients. Little is known about lysozyme in the paranasal sinuses (13)(14)(15)(16) and this study was conceived to determine the expression of lysozyme in the sinus mucosa of normal controls and CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps. ...
... Electron microscopy investigations of inferior turbinate tissue identified lysozyme in serous cells of glands of the nasal mucosa (26) and also in the goblet cells of the nasal epithelium (16) . An immunohistochemical study using nasal turbinate tissue obtained from patients undergoing turbinectomies for nasal obstruction found lysozyme present in serous cells but not in mucous cells or epithelial goblet cells (15) . Our findings in normal sinus specimens are consistent with Fukami et al., (13) where the epithelium was generally lysozyme negative with a weak positive reaction in the goblet cells, and low intensity staining of some serous cells of the subepithelial glands and inflammatory cells throughout the tissue. ...
Background: The presence of fungi and bacteria in the paranasal sinuses may contribute to ongoing inflammation. Lysozyme is an innate immune peptide with bactericidal and fungicidal activity. The expression of lysozyme in chronic rhinosinusitis (CRS) is poorly understood and deficiencies in lysozyme expression may contribute to the ongoing inflammation in CRS patients. Objective: Determine lysozyme expression in sinus mucosa of normal and CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps. Methodology: Sinus mucosa specimens (n = 82) were processed for standard histology, immunohistochemical localisation of lysozyme, immunofluorescent localisation of fungi, and qPCR analysis of lysozyme expression. Results: CRS specimens displayed high-levels of lysozyme immunoreactivity in many of the abundant serous cells. Moderate levels were detected in some epithelial cells and inflammatory cells. Low levels were detected in some subepithelial glands of control specimens. No difference in immunoreactivity was detected between CRSwNP and CRSsNP specimens. Fungal elements were not visualised in any sinus specimen. qPCR analysis demonstrated variable lysozyme expression between individuals. Conclusions: Lysozyme protein expression is increased in patients with CRS, suggesting a defect in lysozyme expression is not responsible for the microbial colonisation often associated with CRS. The functional activity of lysozyme in CRS patients needs to be further investigated.
... However, we have recently demonstrated in vitro that fungi isolated from the mucin of CRS patients were not resistant to lysozyme (9) and hypothesised that a deficiency in lysozyme mRNA expression and/or protein secretion may be present in CRS patients. Little is known about lysozyme in the paranasal sinuses (13)(14)(15)(16) and this study was conceived to determine the expression of lysozyme in the sinus mucosa of normal controls and CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps. ...
... Electron microscopy investigations of inferior turbinate tissue identified lysozyme in serous cells of glands of the nasal mucosa (26) and also in the goblet cells of the nasal epithelium (16) . An immunohistochemical study using nasal turbinate tissue obtained from patients undergoing turbinectomies for nasal obstruction found lysozyme present in serous cells but not in mucous cells or epithelial goblet cells (15) . Our findings in normal sinus specimens are consistent with Fukami et al., (13) where the epithelium was generally lysozyme negative with a weak positive reaction in the goblet cells, and low intensity staining of some serous cells of the subepithelial glands and inflammatory cells throughout the tissue. ...
... To be able to relate the results from this study to studies on other parameters of the innate immune defense, we initially determined the concentrations of lysozyme, a key effector molecule of the protein-mediated arm of innate defense, in nasal fluid. Lysozyme concentrations measured in this study agree with previously published ranges of lysozyme concentrations in nasal secretions (5,12,25). Our measurements reveal a significant increase in lysozyme concentrations in both active and inactive subjects after exercise. ...
... Subsequently, albumin concentration in the nasal fluid samples was assessed as an indicator of plasma transudation. Previous, nonexercise investigations of albumin reported values similar to resting levels in the present study after considering the wash volumes applied in the nasal fluid collection (3,25). Browning et al. (3) studied approximately the same number of subjects and reported similar variance in the individual concentrations of albumin (T 0.1%) as the present study. ...
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 (CEs) have emerged as important effector molecules of innate defense against infections. Here, we compared antimicrobial CEs in nasal fluid before and after moderate-to-vigorous exercise between active and inactive subjects.
Nasal fluid was collected from fourteen healthy, recreationally-active subjects (32 ± 11 yr, 7 males, 7 females) and 14 healthy, inactive subjects (25 ± 3 yr, 7 males, 7 females) 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.
Baseline concentrations (means ± SEM, inactive vs. active) of lysozyme (117.7±31.1 μg/mL vs. 122.9±15.5 μg/mL), CL+CA (15.3±1.8 μg/mL vs.26.2±10.05 μg/mL), and albumin (156.6± 54.5μg/mL vs.126.9±32.8 μg/mL) were similar to previously reported levels and did not differ significantly between study groups. However, post-exercise, CL+CA concentration was significantly lower in inactive compared to active subjects (7.8 ± 1.5 μg/mL vs. 20.1 ± 4.8 μg/mL, p = 0.036) dropping below the antimicrobial effective range. Once adjusted to albumin concentrations the changes were no longer significant, suggesting that plasma transudation accounted for the increased CA+CL concentration post-exercise in the active group relative to the inactive group.
Moderate-to-vigorous aerobic exercise acutely decreases the antimicrobial CE response in inactive subjects, but does not modify baseline levels of CEs between active and inactive subjects. This suggests that compared to active individuals, inactive individuals may be at greater risk for URI immediately post-exercise.
... The secretory cells of the submucosal gland of the nasal cavity are composed of serous cells and mucous cells. The water-rich secretions produced by serous cells consist of proteins and peptides such as lysozyme, lactoferrin, and sIgA, which play various antimicrobial roles [66]. On the other hand, mucous cells secrete mucin, a highly glycosylated protein. ...
The pathophysiology of CRS is multifactorial and complex yet needs to be completed. Recent evidence emphasizes the crucial part played by epithelial cells in the development of CRS. The epithelial cells act as physical barriers and play crucial roles in host defense, including initiating and shaping innate and adaptive immune responses. This review aims to present a comprehensive understanding of the significance of nasal epithelial cells in CRS. New research suggests that epithelial dysfunction plays a role in developing CRS through multiple mechanisms. This refers to issues with a weakened barrier function, disrupted mucociliary clearance, and irregular immune responses. When the epithelial barrier is compromised, it can lead to the passage of pathogens and allergens, triggering inflammation in the body. Furthermore, impaired mucociliary clearance can accumulate pathogens and secretions of inflammatory mediators, promoting chronic inflammation. Epithelial cells can release cytokines and chemokines, which attract and activate immune cells. This can result in an imbalanced immune response that continues to cause inflammation. The interaction between nasal epithelial cells and various immune cells leads to the production of cytokines and chemokines, which can either increase or decrease inflammation. By comprehending the role of epithelial cells in CRS, we can enhance our understanding of the disease’s pathogenesis and explore new therapeutics.
... Perhaps Rothia produce their own cationic peptides that act to permeabilize the outer membrane to SagA, or take advantage of a host-produced protein in vivo. In the latter case, one such possibility is lactoferrin, which is known to permeabilize the outer membrane of Gram-negative bacteria (71) and is present at high concentrations within nasal secretions (72). Therefore, further work is required to determine how SagA gains access to the peptidoglycan of M. catarrhalis. ...
Moraxella catarrhalis is found almost exclusively within the human respiratory tract. This pathobiont is associated with ear infections and the development of respiratory illnesses, including allergies and asthma. Given the limited ecological distribution of M. catarrhalis, we hypothesized that we could leverage the nasal microbiomes of healthy children without M. catarrhalis to identify bacteria that may represent potential sources of therapeutics. Rothia was more abundant in the noses of healthy children compared to children with cold symptoms and M. catarrhalis. We cultured Rothia from nasal samples and determined that most isolates of Rothia dentocariosa and "Rothia similmucilaginosa" were able to fully inhibit the growth of M. catarrhalis in vitro, whereas isolates of Rothia aeria varied in their ability to inhibit M. catarrhalis. Using comparative genomics and proteomics, we identified a putative peptidoglycan hydrolase called secreted antigen A (SagA). This protein was present at higher relative abundance in the secreted proteomes of R. dentocariosa and R. similmucilaginosa than in those from non-inhibitory R. aeria, suggesting that it may be involved in M. catarrhalis inhibition. We produced SagA from R. similmucilaginosa in Escherichia coli and confirmed its ability to degrade M. catarrhalis peptidoglycan and inhibit its growth. We then demonstrated that R. aeria and R. similmucilaginosa reduced M. catarrhalis levels in an air-liquid interface culture model of the respiratory epithelium. Together, our results suggest that Rothia restricts M. catarrhalis colonization of the human respiratory tract in vivo. IMPORTANCE Moraxella catarrhalis is a pathobiont of the respiratory tract, responsible for ear infections in children and wheezing illnesses in children and adults with chronic respiratory diseases. Detection of M. catarrhalis during wheezing episodes in early life is associated with the development of persistent asthma. There are currently no effective vaccines for M. catarrhalis, and most clinical isolates are resistant to the commonly prescribed antibiotics amoxicillin and penicillin. Given the limited niche of M. catarrhalis, we hypothesized that other nasal bacteria have evolved mechanisms to compete against M. catarrhalis. We found that Rothia are associated with the nasal microbiomes of healthy children without Moraxella. Next, we demonstrated that Rothia inhibit M. catarrhalis in vitro and on airway cells. We identified an enzyme produced by Rothia called SagA that degrades M. catarrhalis peptidoglycan and inhibits its growth. We suggest that Rothia or SagA could be developed as highly specific therapeutics against M. catarrhalis.
... They can be produced by AECs, neutrophils, and macrophages [111]. In the early histochemical analysis of turbinate tissue, it is indicated that LYZ and LTF are present in the plasma cells of the submucosal glands [112]. The report proposes that LYZ hydrolyzes bacterial cell walls and modifies mucus viscoelasticity in the nasal epithelium [113,114]. ...
As the first line of defense against risk factors, the nasal epithelial barrier maintains homeostasis in nasal mucosa. The composition of the epithelial barrier contains physical, chemical, immune, and microbiological barriers. Together, these barriers form the nasal defense against irritations. Risk factors from both internal and external environments can disrupt them. External risk factors contain allergens containing proteases, bacteria, virus, particulate matter, diesel exhaust particles, and cigarette smoke. In the meantime, inflammatory cytokines also increase the load on the barrier. Taking into account the role of the epithelial barrier in the nasal mucosa, some studies focus on the treatment of allergic rhinitis (AR) and chronic rhinosinusitis (CRS) by restoring the epithelial barrier, and some progress has been made. Among the therapeutic approaches, histone deacetylase (HDAC) inhibitor and steroid corticosteroids are considered two of the more studied categories, and their roles in repairing barriers have been demonstrated in AR and CRS. The underlying mechanism of HDAC inhibitor may be related to the transcription factor p63. And the protection of corticosteroids may be associated with the allergic disease susceptibility gene, protocadherin-1. Notably, manipulation of the microbiological barrier also has a positive effect on AR and CRS. Lactococcus and probiotics are two categories that are worth being explored continuously. We here review and discuss the compositions and risk factors of the nasal epithelial barrier. Furthermore, some novel and promising approaches to restore the defective barrier in nasal allergic diseases were mentioned.
... LF, produced by serous cells of the submucosal glands, is present in nasal secretions [62], where its concentration increases rapidly after provocation with methacholine, histamine, or allergen [63,64]. It was suggested that LF could be a biomarker for the early detection of allergic rhinitis, as serum LF concentration combined with antigen-specific IgE levels predicted allergic rhinitis with a sensitivity and specificity of 76% and 79%, respectively [65]. ...
Lactoferrin (LF) is a multifunctional iron-binding glycoprotein that exhibits a variety of properties, such as immunomodulatory, anti-inflammatory, antimicrobial, and anticancer, that can be used to treat numerous diseases. Lung diseases continue to be the leading cause of death and disability worldwide. Many of the therapies currently used to treat these diseases have limited efficacy or are associated with side effects. Therefore, there is a constant pursuit for new drugs and therapies, and LF is frequently considered a therapeutic agent and/or adjunct to drug-based therapies for the treatment of lung diseases. This article focuses on a review of the existing and most up-to-date literature on the contribution of the beneficial effects of LF on the treatment of lung diseases, including asthma, viral infections, cystic fibrosis, or lung cancer, among others. Although in vitro and in vivo studies indicate significant potency of LF in the treatment of the listed diseases, only in the case of respiratory tract infections do human studies seem to confirm them by demonstrating the effectiveness of LF in reducing episodes of illness and shortening the recovery period. For lung cancer, COVID-19 and sepsis, the reports are conflicting, and for other diseases, there is a paucity of human studies conclusively confirming the beneficial effects of LF.
... Journal of Immunology Research lysozyme and lactoferrin [40], or chemical defences, such as uric acid, in the epithelial lining fluid [41]. Specific immune functions facilitate the coupling of IgA with the secretory component-specific defence system, which consists of immunological humoral and cellular responses. ...
Allergic rhinitis is a global illness that puzzles many researchers. Most patients with allergic rhinitis also have lower airway hyperresponsiveness, and an allergic rhinitis attack can increase lower airway hyperresponsiveness. However, the mechanism of the effect of allergic rhinitis on the lower airways is still unclear. In this paper, the effects of allergic rhinitis on the lower airways are studied in terms of epidemiology, anatomy, pathophysiology, nasal function loss, inflammation drainage, nasobronchial reflex, and whole-body circulatory flow to determine the mechanism involved and provide ideas for future diagnosis, treatment, and experiments.
... Studies describing the nutritional composition of human nasal secretions have focused on healthy human volunteers [13,99]. However, little attention has been paid to the nutritional status of the volunteers. ...
The human nasal microbiome can be a reservoir for several pathogens, including Staphylococcus aureus. However, certain harmless nasal commensals can interfere with pathogen colonisation, an ability that could be exploited to prevent infection. Although attractive as a prophylactic strategy, manipulation of nasal microbiomes to prevent pathogen colonisation requires a better understanding of the molecular mechanisms of interaction that occur between nasal commensals as well as between commensals and pathogens. Our knowledge concerning the mechanisms of pathogen exclusion and how stable community structures are established is patchy and incomplete. Nutrients are scarce in nasal cavities, which makes competitive or mutualistic traits in nutrient acquisition very likely. In this review, we focus on nutritional interactions that have been shown to or might occur between nasal microbiome members. We summarise concepts of nutrient release from complex host molecules and host cells as well as of intracommunity exchange of energy-rich fermentation products and siderophores. Finally, we discuss the potential of genome-based metabolic models to predict complex nutritional interactions between members of the nasal microbiome.
... [129,130] Nasal lavage with saline Cytology Manual or automated counting of eosinophil influx post-nasal allergen challenge; shown to increase post allergen challenge and differentiate between allergic rhinitics and controls [5,134] Nasal lining fluid Mediators Direct sampling with absorbent strips or sponges produces concentrated sample of lower volume [121,135] Nasal lining fluid Tryptase ng/mL Highly stable, measured by ImmunoCAP; peaks 5-10 minutes after nasal challenge. [121,136] Nasal lining fluid Histamine ng/mL Less stable than tryptase, elevated during early phase response, may be second peak during late phase response [137] Nasal lining fluid Cysteinyl leukotrienes pg/mL Elevated during early phase response; level correlated with clinical symptom scores after nasal allergen challenge [138] Nasal lining fluid T2 cytokines pg/mL Measurable by commercial multiplex immunoassay; IL-4, IL-5, IL-13 elevated from 6 hours post grass pollen nasal allergen challenge, inhibited by allergen immunotherapy [121,135] Nasal lining fluid T2 chemokines pg/mL Eotaxin, RANTES elevated from 4-6 hours post allergen challenge [130,139] Nasal lining fluid Eosinophil activation markers ng/mL ECP (measurable by ImmunoCAP), MBP, EDN; elevated at 6-8 hours post allergen challenge; also reported at 24 hours [140,141] Nasal lining fluid Neuropeptides pg/mL Substance P, CGRP, VIP increased during the early phase reaction [142] Nasal lining fluid Markers of plasma leakage/transudate µg/mL Alpha-2-macroglobulin, albumin elevated in early and late phase response to allergen [143,144] Nasal lining fluid Markers of glandular secretion µg/mL Lactoferrin, lysozyme [145] Nasal lining fluid Fluid metabolomics Differences in metabolic pathways and metabolite levels seen in patients with N-ERD versus controlsyet to be studied in allergics post allergen challenge [146] Nasal cytology brushing Nasal mucosal gene expression Nasal scrapes 8 hours post allergen challenge and environmental exposure chamber show similar pattern of altered expression of mucosal biology and transcriptional regulation genes; nasal ACE2 receptor expression reduced after allergen challenge. [147][148][149] Nasal curettage Cytology Nasal curettage 24 hours post allergen challenge show increases in mucosal ILC2 cells [150] Nasal biopsy Cytology Compartmental assessment of allergen-induced inflammatory cells. ...
Allergen provocation test is an established model of allergic airway diseases, including asthma and allergic rhinitis, allowing the study of allergen-induced changes in respiratory physiology and inflammatory mechanisms in sensitised individuals as well as their associations. In the upper airways, allergen challenge is focused on the clinical and pathophysiological sequelae of the early allergic response and applied both as a diagnostic tool and in research settings. In contrast, the bronchial allergen challenge has almost exclusively served as a research tool in specialised research settings with a focus on the late asthmatic response and the underlying type 2 inflammation. The allergen-induced late asthmatic response is also characterised by prolonged airway narrowing, increased non-specific airway hyperresponsiveness and features of airway remodelling including the small airways, and hence, allows the study of several key mechanisms and features of asthma. In line with these characteristics, the allergen challenge has served as a valued tool to study the crosstalk of the upper and lower airways and in proof of mechanism studies of drug development. In recent years, several new insights into respiratory phenotypes and endotypes including the involvement of the upper and small airways, innovative biomarker sampling methods and detection techniques, refined lung function testing as well as targeted treatment options, further shaped the applicability of the allergen provocation test in precision medicine. These topics, along with descriptions of subject populations and safety, in line with the updated GINA2021, will be addressed in this paper.
... Lactoferrin is an iron-binding glycoprotein that limits bacterial growth through sequestration of iron and also exerts direct antibacterial activity [10][11][12][13] . Studies identified a synergistic effect of lactoferrin and lysozyme in enhancing bacterial killing of gram-negative bacteria [14][15][16] . In addition to numerous antimicrobial peptides, SIgA contributes significantly to first-line defenses at mucosal sites 17 . ...
Young age is a risk factor for respiratory and gastrointestinal infections. Here, we compared infant and adult mice to identify age-dependent mechanisms that drive susceptibility to mucosal infections during early life. Transcriptional profiling of the upper respiratory tract (URT) epithelium revealed significant dampening of early life innate mucosal defenses. Epithelial-mediated production of the most abundant antimicrobial molecules, lysozyme, and lactoferrin, and the polymeric immunoglobulin receptor (pIgR), responsible for IgA transcytosis, was expressed in an age-dependent manner. This was attributed to delayed functional development of serous cells. Absence of epithelial-derived lysozyme and the pIgR was also observed in the small intestine during early life. Infection of infant mice with lysozyme-susceptible strains of Streptococcus pneumoniae or Staphylococcus aureus in the URT or gastrointestinal tract, respectively, demonstrated an age-dependent regulation of lysozyme enzymatic activity. Lysozyme derived from maternal milk partially compensated for the reduction in URT lysozyme activity of infant mice. Similar to our observations in mice, expression of lysozyme and the pIgR in nasopharyngeal samples collected from healthy human infants during the first year of life followed an age-dependent regulation. Thus, a global pattern of reduced antimicrobial and IgA-mediated defenses may contribute to increased susceptibility of young children to mucosal infections.
... The distal SMGs consist of two distinct secretory cell types, serous cells, and mucous cells surrounded by contractile myoepithelial cells. Serous cells produce watery fluid containing a diverse of antimicrobial defense molecules including lysozyme, lactoferrin, and sIgA [4]. On the other hand, mucous cells secrete highly viscous mucus rich in mucins such as MUC5B [5,6]. ...
Mucus secretion and its composition are vital in the maintenance of airway health, among which hypoxia-inducible factors (HIFs) are thought to be involved in the regulation of mucin synthesis and regulation. Nasal mucus composition difference between healthy individuals and chronic rhinosinusitis (CRS) patients may contribute to the pathology of chronic nasal diseases, but so far, their role has yet to be completely understood. Nasal biopsy specimens were obtained from 24 healthy subjects and 99 patients with CRS without (CRSsNP, n=36) or with (CRSwNP, n=63) nasal polyps. Immunohistochemical (IHC) and immunofluorescent (IF) staining, quantitative real-time PCR, and western blot were performed to compare the nasal mucus composition between the subjects. Areas of the serous gland and mucous gland were both significantly increased in CRSsNP patients. In CRSwNP patients, a decrease in submucosal gland density and a marked increase in goblet cells were observed. The major gel-forming mucins in the sinonasal mucosa of CRSsNP and CRSwNP are MUC5B and MUC5AC respectively. Mucous cells are found in a higher proportion in both CRSsNP and CRSwNP. The proportion of MUC5AC-positive goblet cells was increased in CRSwNP. The mRNA level of HIF-2α was significantly increased in CRS, and both HIF-1α and HIF-2α were expressed in serous cell but not mucous cell. Over secretion and altered composition of mucus are observed in sinonasal mucosa of CRS, which was mainly associated with glandular hyperplasia in CRSsNP and goblet cell hyperplasia in CRSwNP. Mucus abnormality compromised both non-specific and specific antimicrobial capabilities in the sinonasal mucosa. HIF expression may contribute to differences in mucin synthesis and serous gland regulation, which needs further investigation to understand the pathology of CRS.
... Human respiratory droplets are composed mainly of water (∼ 90 to 99%), with the remainder being mostly inorganic ions, sugars, proteins, lipids, DNA, and, potentially, pathogens. While the exact droplet composition depends strongly on many factors, the typical mass or volume proportion of non-water content in a droplet is around φ 0 = 1 to 10% [48,55,57,77,78]. A hypothetically completely dried-out droplet devoid of water would thus have a radius of: ...
A large number of infectious diseases are transmitted by respiratory droplets. How long these droplets persist in the air, how far they can travel, and how long the pathogens they might carry survive are all decisive factors for the spread of droplet-borne diseases. The subject is extremely multifaceted and its aspects range across different disciplines, yet most of them have only seldom been considered in the physics community. In this review, we discuss the physical principles that govern the fate of respiratory droplets and any viruses trapped inside them, with a focus on the role of relative humidity. Importantly, low relative humidity—as encountered, for instance, indoors during winter and inside aircraft—facilitates evaporation and keeps even initially large droplets suspended in air as aerosol for extended periods of time. What is more, relative humidity affects the stability of viruses in aerosol through several physical mechanisms such as efflorescence and inactivation at the air-water interface, whose role in virus inactivation nonetheless remains poorly understood. Elucidating the role of relative humidity in the droplet spread of disease would permit us to design preventive measures that could aid in reducing the chance of transmission, particularly in indoor environment.
... Human respiratory droplets are composed mainly of water (∼ 90% to 99%), with the remainder being mostly inorganic ions, sugars, proteins, lipids, and DNA. The exact composition, however, strongly depends on the type and the location of secretion [48,[55][56][57][58]. Table 1 shows an overview of the basic components measured in droplets of different origins, such as respiratory fluid and saliva. ...
A large number of infectious diseases is transmitted by respiratory droplets. How long these droplets persist in the air, how far they can travel, and how long the pathogens they might carry survive are all decisive factors for the spread of droplet-borne diseases. The subject is extremely multifaceted and its aspects range across different disciplines, yet most of them have only seldom been considered in the physics community. In this review, we discuss the physical principles that govern the fate of respiratory droplets and any viruses trapped inside them, with a focus on the role of relative humidity. Importantly, low relative humidity---as encountered, for instance, indoors during winter and inside aircraft---facilitates evaporation and keeps even initially large droplets suspended in air as aerosol for extended periods of time. What is more, relative humidity affects the stability of viruses in aerosol through several physical mechanisms such as efflorescence and inactivation at the air-water interface, whose role in virus inactivation nonetheless remains poorly understood. Elucidating the role of relative humidity in the droplet spread of disease would permit us to design preventive measures that could aid in reducing the chance of transmission, particularly in indoor environment.
... Nano-vehicles performed advantages for enhancing solubility of hydrophobic therapeutic agents and increasing drug accumulation at lesion areas [18] To improve the nasal bioavailability of hydrophobic drugs, the nano-vehicles should possess the property of stimuliresponsive drug release profiles to unique nasal environment (pH 5.5, 33°C, 30 μg/mL lysozyme) [19][20][21]. With large amounts of histamine in nasal secretions of atopic patients, the capacity of burst release of embedded antagonists to relieve allergic symptoms quickly was indispensable. ...
Allergic rhinitis (AR) was a chronic airway inflammatory disease. Nasal administration showed superiorities due to its effective drug absorption. Cetirizine (CTZ) was a common H1-antihistamine in allergic disorders therapy, while hydrophobicity and irritation to nasal mucosa limited its application. In this regard, deoxycholate-chitosan-hydroxybutyl nanoparticles with CTZ covalently grafted and free CTZ encapsulated (CTZ:CDHBCs-NPs) were synthesized as nasal adaptive nano-drug delivery systems. CDHBCs-NPs with various lower critical solution temperature (LCST) (29, 33, 37 °C) were prepared, with particle sizes of ~120 nm and zeta potentials of ~4 mV. In nasal condition (pH 5.5, 33 °C), the diameters of CDHBCs-NPs increased slightly (~129 nm to ~134 nm) because of the pH-responsive expansion. Burst release of free CTZ from CDHBC-29-NPs (~76%) was significantly (p < 0.05) accelerated compared with that of CDHBC-33-NPs and CDHBC-37-NPs (~60%), owing to thermo sensitive drug squeeze out (T > LCST). Incubating with lysozyme (30 μg/mL), CDHBCs-NPs swelled and exhibited ~2-fold increase (p < 0.01) of sizes, with additional CTZ releasing (~5%) attributing to the digestion of polysaccharide backbone covalent connected with CTZ. It could be speculated that stimuli-responsive CDHBCs-NPs might hold tremendous potential as nasal adaptive delivery vehicles in allergic airway inflammatory diseases therapy.
... Identified clusters were present across all patients within the non-polyp and polyp groups Table 3), and display lineage-defining genes to highlight the major cell types recovered: basal 7,8 (KRT5) and apical 7,8 (KRT8) to orient the major division present in pseudostratified epithelia of the respiratory tract, and further specialization present in ciliated 7,9 (FOXJ1) and glandular 10,11 (LTF) apical epithelium. We also highlight the supportive endothelial 12 (DARC), fibroblast 13 (COL1A2), plasma 14 (CD79A), myeloid 15,16 (HLA-DRA), T 17,18 (TRBC2), and mast cells 19 (TPSAB1) (Extended Data Fig. 1e). ...
... Proinflammatory cytokines, such as TNF-α, IL-1β, and IL-8, are potent multifunctional cytokines that play an important role in the pathogenesis of many inflammatory diseases [27]. MUC5AC and LTF are markers of glandular secretion of the nasal mucosa [28,29]. Neuropeptides, such as CGRP and substance P, mediate neurogenic inflammation [30]. ...
Objectives:
Regenerative treatment using stem cells may serve as treatment option for empty nose syndrome (ENS), which is caused by the lack of turbinate tissue and deranged nervous system in the nasal cavity. We aimed to assess the efficacy and safety of the autologous stromal vascular fraction (SVF) in the treatment of ENS.
Methods:
In this prospective observational clinical study, we enrolled 10 ENS patients who volunteered to undergo treatment of ENS through the injection of autologous SVF. Data, including demographic data, pre- and postoperative Sino-Nasal Outcome Test-25 (SNOT-25) scores, overall patient satisfaction, and postoperative complications, were prospectively collected. Nasal secretion was assessed using the polyurethane foam absorption method, and the levels of biological markers were analyzed in both ENS group and control group using enzyme-linked immunosorbent assay. The SVF extracted from abdominal fat was diluted and injected into both inferior turbinates.
Results:
Among the 10 initial patients, one was excluded from the study. Subjective satisfaction was rated as "much improved" in two and "no change" in seven. Among the improved patients, the mean preinjection SNOT-25 score was 55.0 and the score at 6 months after injection was 19.5. However, the average SNOT-25 score of nine participants at 6 months after injection (mean±standard deviation, 62.4±35.8) did not differ significantly from the baseline SNOT25 score (70.1±24.7, P>0.05, respectively). Among the various inflammatory markers assessed, the levels of interleukin (IL)-1β, IL-8, and calcitonin gene-related peptide were significantly higher in ENS patients. Compared with preinjection secretion level, the nasal secretions from SVF-treated patients showed decreased expressions of IL-1β and IL-8 after injection.
Conclusion:
Although SVF treatment appears to decrease the inflammatory cytokine levels in the nasal mucosa, a single SVF injection was not effective in terms of symptom improvement and patient satisfaction. Further trials are needed to identify a more practical and useful regenerative treatment modality for patients with ENS.
... Immunoglobulin A (IgA) is the predominant immunoglobulin present on nasal mucosal surfaces, in which it acts as an active barrier against inhaled antigens and pathogens [27]. Secretory IgA (sIgA) is produced by plasma cells locally in the nasal mucosa and can be used as a marker of glandular secretions [28]. It takes part in the mucosal immunity of target organs of allergic diseases, such as the nasal airways [29]. ...
Purpose of review:
The identification of immunological markers in nasal secretions and tears is becoming essential in the study of allergic diseases. The collection procedure of nasal and ocular secretions directly influences the results, thus it is of paramount importance to validate and standardize the sampling process.
Recent findings:
Current techniques for nasal secretions sampling are mainly based on three principles: collection of spontaneous secretions, nasal washings, and absorption. Collection of spontaneous secretions is appropriate in subjects with nasal hypersecretion, whereas in healthy individuals the collected volume is frequently insufficient. Nasal washings are associated with an unpredictable, high dilution and concentrations of markers often fall below detection limits of immunological assays. Absorption seem to provide the best compromise between sufficient sample amounts and detectability of inflammatory mediators and immunoglobulin E. Tear samples can be obtained by glass capillary tubes, filter paper strips and ophthalmic sponges. Volumes are however small or highly diluted through reflex tearing.
Summary:
Secretions reflect the local inflammatory activity and provide valuable information about the immunological reaction to allergens at the target organ. There is increasing evidence of the potential clinical role of their analysis, for diagnosis, and monitoring of allergic rhino-conjunctivitis. Appropriate collection and processing is very important and requires special attention.
... Because many innate defense proteins secreted by the airway epithelium act in part through bacterial lysis or permeabilization (e.g. lysozyme [62][63][64][65][66][67] and β-defensins [68][69][70][71]), we sought to test whether flavones may enhance the efficacy of airway antimicrobials. We tested the effects of flavones combined with the important airway antimicrobial lysozyme, which is secreted by airway submucosal gland serous cells [72][73][74] and catalyzes the hydrolysis of 1,4-beta-linkages between N-acetyl-D-glucosamine and N-acetylmuramic acid residues of peptidoglycan in the bacterial cell wall [75,76]. ...
Flavones are a class of natural plant secondary metabolites that have anti-inflammatory and anti-bacterial effects. Some flavones also activate the T2R14 bitter taste receptor, which is expressed in motile cilia of the sinonasal epithelium and activates innate immune nitric oxide (NO) production. Flavones may thus be potential therapeutics for respiratory infections. Our objective was to examine the anti-microbial effects of flavones on the common sinonasal pathogens Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa, evaluating both planktonic and biofilm growth. Flavones had only very low-level antibacterial activity alone. They did not reduce biofilm formation, but did reduce production of the important P. aeruginosa inflammatory mediator and ciliotoxin pyocyanin. However, flavones exhibited synergy against P. aeruginosa in the presence of antibiotics or recombinant human lysozyme. They also enhanced the efficacy of antimicrobials secreted by cultured and primary human airway cells grown at air-liquid interface. This suggests that flavones may have anti-gram-negative potential as topical therapeutics when combined with antibiotics or in the context of innate antimicrobials secreted by the respiratory or other epithelia. This may have an additive effect when combined with T2R14-activated NO production. Additional studies are necessary to understand which flavone compounds or mixtures are the most efficacious.
... Our results show that lysozyme at physiological concentrations [23,46] has a very modest antipseudomonal activity. However, when the permeability barrier is overpassed (through subinhibitory concentrations of colistin), its bactericidal activity is greatly enhanced. ...
Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our results help to delineate new strategies against P. aeruginosa infections, simultaneously targeting β–lactam resistance, cell-wall metabolism and virulence, ultimately enhancing the activity of our innate immune weapons.
... Therefore, it is highly unlikely that the increased lysozyme levels may be due to an active immune response against bacterial infections. It is well known that lysozyme is an index of serous glandular secretions [72,73]. It can thus be speculated that allergic subjects may display hypertrophy or hyper-activation of the nasal submucosal glands. ...
Discriminating different rhinitis cases can sometimes be difficult as the diagnostic criteria used to identify the various subgroups are not always unambiguous. The nasal fluid (NF) highly reflects the pathophysiology of these inflammatory diseases. However, its collection, as nasal lavage fluid (NLF), may cause discomfort. Due to the non-invasiveness and rapidity of collection, nasal swab might represents an alternative to overcome these problems and also an ideal source of biomarkers. In this study we demonstrate that the combined use of mesoporous silica (MPS) with MALDI-TOF MS allows the rapid detection of differential nasal peptide profiles from nasal swabs of healthy (H), allergic rhinitis (AR) and non-allergic rhinitis (NAR) subjects. NF peptides from nasal swabs were captured by the mean of MPS then profiled by MALDI-TOF MS. As a proof-of-principle, we also explored the ability of our platform to discriminate between nasal swabs of patients with AR and NAR, and between these groups and H controls. Four peaks resulted differentially expressed between NAR and AR, two peaks discriminated AR from H while one peak segregated NAR from H group. Therefore, peptides selected and enriched by our platform could form part of a diagnostic ''rhinomic'' profile of the allergic and non-allergic patients. This article is protected by copyright. All rights reserved.
... The majority of lysozymes are secreted molecules and in humans the c-type lysozyme is found in various body fluids, such as tears, saliva, airway mucus, breast milk, urine, serum, cerebrospinal fluid, cervical mucus, and amniotic fluid; in tissues of the respiratory and intestinal tract; and in the lysosomal granules of neutrophils and macrophages (Hankiewicz and Swierczek, 1974;Gordon et al., 1974;Brouwer et al., 1984;Raphael et al., 1989;Borregaard et al., 2007;McDermott, 2013). ...
Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are evolutionarily conserved innate immunity molecules homologous to bacteriophage type 2 amidases. Mammalian PGRPs are soluble secreted proteins and bind muramyl peptide fragments of bacterial peptidoglycan. Mammalian PGLYRP1, PGLYRP3, and PGLYRP4 are directly bactericidal and kill bacteria by inducing an exaggerated envelope stress response, which causes oxidative, thiol, and metal stress, membrane depolarization, inhibition of biosynthetic reactions, and bacterial death. Mammalian PGLYRP2 is an enzyme, peptidoglycan amidohydrolase. In vivo, mammalian PGRPs maintain a healthy gut microbiome, which protects animals from experimental colitis. Mammalian PGRPs also modulate sensitivity to skin and joint inflammation and allergic asthma. Human PGRP variants are associated with sensitivity to inflammatory bowel diseases, psoriasis, and Parkinson's disease. PGRPs in lower vertebrates, echinoderms, and mollusks are usually bactericidal amidases and protect against infections. PGRPs in insects are more numerous and diverse and are the main sensors of bacterial infections. Insect PGRPs (1) induce signaling cascades (Toll and IMD), which trigger production of antimicrobial peptides; (2) induce the enzymatic prophenoloxidase cascade, which generates other antimicrobial products; and (3) downregulate antimicrobial responses by hydrolyzing stimulatory peptidoglycan or by antagonizing cell-activating receptors. Lysozyme is a peptidoglycan-hydrolyzing muramidase present in all animals. Lysozyme has antibacterial activity and eliminates pro-inflammatory peptidoglycan.
... µg/ml, manifesting no significant difference as compared to nasal fluids in carriers of S. aureus. It is difficult to compare the results to those obtained in earlier studies (Raphael et al., 1989;Noble, 2002), the authors of which estimated Ly activity using a turbidimetric assay, based on the enzymatic hydrolysis of bacterial cell walls. Therefore, the obtained data allow to conclude that Ly does not prevent against nasal carriage of S. aureus. ...
Nasal carriage of Staphylococcus aureus represents a well-defined factor of risk involving community and hospital-acquired infections. Recently a significance of several host factors has been pointed out and, in particular, of immune determinants in nasal S. aureus colonization. Therefore, this study aimed at analysis of manifestation involving manifestation in the nasal secretions of important components of the host innate immunity – human beta-defensin-2 (HBD-2), lysozyme (Ly), and interferon-gamma (IFN-γ) in healthy individuals and in persons with persistent carriage of S. aureus. The studies were conducted in two groups of healthy volunteers, encompassing non-carriers (group 1) or persistent carriers of S. aureus (group 2). Elisa assays were employed to evaluate levels of HBD-2, Ly, and IFN-γ in nasal secretions of the examined donors. In S. aureus carriers a significant variability of HBD-2 levels was detected, corresponding to, respectively, the high (averaging at 1.46 ng/ml) and the low (averaging at 0.13 ng/ml) secretory response of the defensin. The level of Ly in S. aureus carriers averaged at 1.46 μg/ml and it manifested no significant difference as compared to that noted in non-carriers. In turn, concentrations of IFN-γ in nasal secretions in the group of carriers of S. aureus amounted on the average to 81.7 pg/ml and they were 1.3-fold higher that in the group of non-carriers. The obtained results allow to conclude that IFN-γ secretion by the nasal cavity-colonizing S. aureus remains quantitatively insufficient to eliminate the pathogen. Nevertheless, a significant increase in levels of this host factor may be important for restriction of the staphylococcal colonization and protection against development of an invasive infection. In turn, the role of HBD-2 and Ly in inactivation of the colonizing S. aureus remains doubtful.
... A possible role of nasal mucus could be to block pollen proteases with innate antiproteases and to prevent subsequent epithelial damage and allergen transport through the mucosa [6][7][8][9]. Proteins exudated from plasma could have immunomodulatory functions and influence disease severity [10,11]. To understand the nasal mucus proteome proteomic studies are feasible detecting a large range of proteins and facilitating analysis of their origin and function [12]. ...
Introduction:
Nasal mucus and its proteins are a defence against allergens. We sought to investigate dynamic proteome changes in allergic rhinitis upon environmental allergen provocation.
Methods:
Nasal mucus was collected in and out of pollen season from allergic rhinitis patients (N=10) and healthy controls (N=12). Liquid chromatography-tandem mass spectrometry was performed. Proteins were identified by SwissProt database search and quantified from normalized areas under curve of precursor ion chromatograms. Gene enrichment analysis was performed with Cytoscape/BINGO software.
Results:
In total 430 different proteins were detected in both groups, 203 (47.2%) were newly identified. In allergics CLU and IGKC were significantly more abundant in season (2.2 and 2.1-fold respectively). GSTP1 (0.5-fold), ELANE (0.4-fold), HIST1H2BK (0.3-fold), S100A8 (0.2-fold), S100A12 (0.2-fold) and ARHGDIB (0.1-fold) were significantly less abundant in season. In healthy controls UBC, TUBA1B, HBB and FABP5 were only present in season. Ig kappa chain V-I region DEE (5.3-fold), CLU (5.0-fold), TXN (4.3-fold), MSMB (3.2-fold) and Ig heavy chain V-III region BRO (2.7-fold) were significantly more abundant in season. MUC5B (0.5-fold), SLPI (0.2-fold) and S100P (0.2-fold) were significantly less abundant in season.
Conclusion:
Contrary to their symptoms allergic rhinitis patients show perennial inflammatory response lacking adequate reaction to allergens in season.
Biological significance:
Many studies dealing with allergic rhinitis are focused on the nasal epithelium. This is the first study to analyse the nasal mucus as primary defence barrier on a proteomic level in and out of pollen season and contrary to the leading opinion shows that allergic patients show a perennial inflammatory response with reduced reaction to allergens whereas healthy controls react on proteome basis towards enhanced defence in season despite lacking allergic sensitization.
... 17 The fluid secreted by these cells directly contributes to airway surface liquid (see later) volume, hydration of mucus released from goblet cells, 18,19 and innate immunity. 20 Basal cells are connected to the basement membrane via hemodesmosomes, providing the foundation for the attachment of ciliated and goblet cells to basal lamina, and also have the potential to regulate inflammatory responses, oxidant defense and transepithelial water movement. 21 The most important feature of basal cells is their capacity to repopulate all the major cell types of conductive airway epithelium. ...
Airway epithelium contributes significantly to the barrier function of airway tract. Mucociliary escalator, intercellular apical junctional complexes which regulate paracellular permeability and antimicrobial peptides secreted by the airway epithelial cells are the three primary components of barrier function of airway tract. These three components act cooperatively to clear inhaled pathogens, allergens and particulate matter without inducing inflammation and maintain tissue homeostasis. Therefore impairment of one or more of these essential components of barrier function may increase susceptibility to infection and promote exaggerated and prolonged innate immune responses to environmental factors including allergens and pathogens resulting in chronic inflammation. Here we review the regulation of components of barrier function with respect to chronic airways diseases.
... It must be noted that in addition to their likely role in secreting a primary Cl − -and HCO 3 − -rich fluid, the serous cells of submucosal glands secrete a vast array of antimicrobial peptides (reviewed in [122]) that help neutralize pathogens while they are trapped in the airway mucus. These include lysozyme [123,124], lactoferrin ( [125][126][127][128], lactoperoxidase [129], beta-defensin 2 [19], LL-37 [20,130], and the mucin Muc7 [11]. It is very likely that Ca 2+ signaling regulates both fluid and antimicrobial secretion from serous acinar cells, as well as mucus secretion from mucous cells. ...
Cytoplasmic Ca2+ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca2+ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca2+. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca2+ as a second messenger. Changes in intracellular Ca2+ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca2+-activated K+ channels and Cl− channels. We also review evidence of interactions of Ca2+ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca2+ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.
... Once inside the nasal cavity, these particles are deposited onto the nasal sinus cavity mucosa and undergo mucociliary clearance to the back of the nasopharynx and thence to the oropharynx from where they are excreted via the gastrointestinal tract (29). The presence of cytokines and inflammatory mediators in the mucosa neutralizes infective species (30). ...
To determine the effect of intranasal sodium hyaluronate on mucociliary clearance time following functional endoscopic sinus surgery in patients with nasal polyposis.
Randomized, controlled, blinded study.
Thirty-six patients with grade II nasal polyposis undergoing functional endoscopic sinus surgery received intranasal sodium hyaluronate 9 mg twice daily or saline for 30 days commencing on the second day after surgery. Ciliary mucous transport time was assessed using charcoal powder and saccharin administered during rhinoscopy. Other outcomes included changes in symptoms, endoscopic appearance of the nasal mucosa, and tolerability.
Patients receiving sodium hyaluronate had a significantly faster mucociliary clearance time at 1 month compared with controls (14.3 +/- 2.5 vs. 23.6 +/- 3.3 minutes; p = 0.000). Furthermore, sodium hyaluronate recipients experienced a lower incidence of rhinorrhea, less nasal obstruction and a lower incidence of exudate on endoscopy than control subjects at 1 month (all p < 0.05). Sodium hyaluronate was well tolerated in patients following functional endoscopic sinus surgery.
The use of intranasal sodium hyaluronate in patients undergoing functional endoscopic sinus surgery for nasal polyposis augmented the improvement in mucociliary clearance observed following this procedure and improved several clinical and endoscopic parameters. These data provide encouraging evidence of the beneficial effects of sodium hyaluronate in the care of patients undergoing functional endoscopic sinus surgery with which to continue the development of the product for this indication.
This chapter summarizes the methods used to study the nasal passages in relation to air pollutants such as nasal smears, imprints, and brush. Nasal smears are obtained with cotton-wool swabs gently moved along the anterior to the posterior part of the inferior and/or the middle turbinates. The swab is then either smeared over a glass slide and the specimen is fixed and stained, or it is immersed in a transport fluid for microbiology culture techniques. This method yields cells from nasal secretions and the superficial portion of the nasal mucosa. In imprints method, plastic strips covered with 1% albumin are introduced into the nasal cavity under direct vision and are gently pressed over a nasal mucosa surface. The strips are then fixed, stained, and examined under the microscope. The material obtained contains nasal secretions and superficial epithelium. In brush technique, epithelium is harvested with a small brush made of plastic-coated steel wire with nylon bristles. The brush is introduced into the nasal cavity under direct vision and is rotated while being introduced and removed. The brush is then submerged into a saline-buffered solution or transport media if cell culture is the final choice. The brush is shaken, so the harvested cells go into suspension. The advantages of the procedure are the quantification of the total number of cells per volume by hemocytometer, staining of multiple slides for quantification of different cell types, and the use of cell pellets for biochemical analysis.
Pseudomonas aeruginosa is one of the most important opportunistic pathogens, whose clinical relevance is not only due to the high morbidity/mortality of the infections caused, but also to its striking capacity for antibiotic resistance development. In the current scenario of a shortage of effective antipseudomonal drugs, it is essential to have thorough knowledge of the pathogen's biology from all sides, so as to find weak points for drug development. Obviously, one of these points could be the peptidoglycan, given its essential role for cell viability. Meanwhile, immune weapons targeting this structure could constitute an excellent model to be taken advantage of in order to design new therapeutic strategies. In this context, this review gathers all the information regarding the activity of mammalian peptidoglycan-targeting innate immunity (namely lysozyme and peptidoglycan recognition proteins), specifically against P. aeruginosa. All the published studies were considered, from both in vitro and in vivo fields, including works that envisage these weapons as options not only to potentiate their innate effects within the host or for use as exogenously administered treatments, but also harnessing their inflammatory and immune regulatory capacity to finally reduce damage in the patient. Altogether, this review has the objective of anticipating and discussing whether these innate immune resources, in combination or not with other drugs attacking certain P. aeruginosa targets leading to its increased sensitization, could be valid therapeutic antipseudomonal allies.
Deux vaccins sont actuellement disponibles pour la prévention des infections invasives à pneumocoques (IIP) : un vaccin polysaccharidique Pneumovax® (PPV23) et un vaccin conjugué Prevenar13® (PCV13), induisant respectivement une protection contre 23 et 13 sérotypes. Le PPV23 est considéré comme faiblement immunogène, en particulier chez les personnes âgées et les patients immunodéprimés. Le PCV13, en revanche, en raison de la conjugaison à une protéine porteuse, présente l'avantage d'induire une réponse immunitaire T-dépendante, non observée avec le vaccin PPV23. Dans notre travail nous avons donc évalué l'impact des stratégies vaccinales utilisant le PCV13 et le PPV23 sur différentes populations de patients à risque. Dans une première étude, nos résultats sur la vaccination anti-pneumococcique chez des patients atteints de myélome indolent (SMM) ont montré qu'une dose de PCV13 seul, induisait une réponse immune transitoire et de faible persistance. Ces résultats suggéraient l'utilisation d'un schéma vaccinal incluant plusieurs doses de PCV13 ou une association avec le PPV23. Depuis 2013, ce schéma combiné du PCV13 et du PPV23 est le schéma recommandé par la Haute Autorité de Santé en France chez les patients à risque, avec les délais suivants : une dose de PCV13 suivie d'une dose de PPV23, 8 semaines après. Nous avons par la suite étudié cette stratégie vaccinale combinée chez des patients à risque d'IIP : patients atteints de lupus érythémateux systémique (SLE) et patients atteints de polyarthrite rhumatoïde (PR). Nos résultats montrent une immunogénicité à court terme de la stratégie combinée, mais une protection qui ne persiste pas au-delà de deux ans. De façon surprenante, les taux d'anticorps 2 ans après la vaccination, sont inférieurs aux taux pré-vaccinaux pour les patients PR. Cet effet délétère du PPV23 sur la réponse vaccinale induite par le PCV13 est appelé hyporéponse. Ce phénomène, observé chez les patients PR, ne se retrouve pas chez les patients SLE dont la vaccination PPV23 a été effectuée plus à distance du PCV13. Ces résultats suggèrent que le schéma vaccinal plus tardif (c'est-à-dire une vaccination par le PPV23 six mois après le PCV13 au lieu de deux mois) inhiberait le phénomène d'hyporéponse. Dans une troisième partie, nous avons comparé différents schéma vaccinaux modulant les doses des vaccins et les délais d'injection chez des volontaires sains mais également dans un modèle murin d'hyporéponse développé au sein du laboratoire. Notre hypothèse était que la modulation du schéma vaccinal utilisant les 2 vaccins pouvait à la fois induire une protection à long terme et prévenir l'hyporéponse. Nos résultats ont montré que l'utilisation d'une dose diminuée de PPV23 ou l'injection concomitante des deux vaccins n'empêchaient pas l'hyporéponse. En revanche, en allongeant le délai entre le PCV13 et le PPV23, le phénomène d'hyporéponse est limité. Des études cliniques chez les patients à risque d'IIP sont nécessaires afin d'évaluer une stratégie combinée tardive, où le PPV23 serait reçu au moins 6 à 12 mois après le PCV13.
In Japanese cedar (Cryptomeria japonica: CJ) pollinosis, release of major allergens (Cry j 1 and Cry j 2) from CJ pollens in the nasal cavity and their incorporation into the nasal mucosa are essential for the onset of the disease. The effect of nasal fluid on the release of major allergens from the pollen grains was studied in vitro.
CJ pollens were suspended in normal saline and nasal fluid solution (NFS), and the ratio of rupture of the pollen grain wall on a slide glass was determined under a light microscope. The concentration of protein eluted in the supernatant of the cedar pollen suspension was determined by modified Lowry's protein assay. The major allergens of CJ in the supernatant were quantified by sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit polyclonal antibody. The antigenicity of the pollen extract was examined by the proliferative response of mouse Cry j 2 specific helper T cell lines.
Both the ratio of rupture of the pollen grains and the concentration of protein in the supernatant significantly increased in the presence of NFS. Elution of the major allergens also increased in the presence of NFS, and the amount of Cry j 1 was larger than Cryj 2. The study on various factors involved in elutionof major allergens suggested that elution is promoted by components of nasal fluid, such as proteins, mutins, and lysozyme, as well as such physicochemical factors as pH and temperature. In conclusion, this study indicates that CJ pollens are in the state that is easy to release the major allergens in the nasal cavity of patients with allergic rhinitis.
Nach Referaten zur Jahreshauptversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Halschirurgie über den Geruchsund Geschmackssinn im Jahre 1975 in Wiesbaden und über die Diagnostik und Therapie der Nebenhöhlenentzündungen 1982 in Bad Reichenhall standen spezielle rhinologische Fragen nicht sehr häufig im Zentrum des Interesses der HNO-Gesellschaft, wenn man die für die Jahrestagung vergebenen Referate als ein Kriterium ansehen möchte. Aktuelle Teilaspekte der Rhinologie wurden nochmals 1987 in Bad Neuenahr mit immunpathologischen und allergologischen Referaten behandelt.
The common cold is the cause of the great majority of respiratory tract infections. It seems well established that this is a viral infection in the upper airways. In the early 1950s symptoms and signs of a common cold were induced in isolated healthy individuals for the first time by nasal inoculation of virus cultured in vitro [11]. There is a widespread traditional belief that the symptoms of the common cold are related to cooling of the body. Although cooling, e.g. of the feet, induces physiological alterations in the nasal mucosa [69,82,130], local (or general) cooling has failed to result in disease in volunteers, or even to make them more prone to catch a common cold infection in controlled experiments [10,66]. Common colds are brief, self-limited infections that do not affect the life expectancy of otherwise healthy and well-fed subjects. However, they can be a threat to patients who are immunocompromised, are malnourished or have severe cardiac or pulmonary disease. Malnourishment is a contributing factor in developing countries where the children affected suffer more persistent airway infections and more complications, mainly bronchopneumonia [106], which may lead to considerable mortality [51]. There are vast social, as well as economic consequences, as illustrated by an annual loss of 100 million working days in the USA where each episode of a common cold costs an estimated $15–40 for medications and diagnostic medical consultations [117].
Lactoferrin and lysozyme are two important, naturally occurring antibacterial proteins found in saliva, nasal secretions, milk, mucus, serum and in the lysosomes of neutrophils and macrophages. Both proteins bind specifically to glucose-modified proteins bearing advanced glycation endproducts (AGEs). Exposure to AGE-modified proteins blocks the bacterial agglutination and bacterial killing activities of lactoferrin and also inhibits the bactericidal and enzymatic activity of lysozyme. Peptide mapping by AGE ligand blot revealed two AGE-binding domains in lactoferrin,and a single AGE-binding domain in lysozyme. None of these AGE-binding domains displayed any significant homology in their primary sequences; however, a common 17-18 amino acid cysteine loop motif (CX15-16C) was identified among them, which we named an ABCD motif (AGE-Binding Cysteine-bounded Domain). Similar domains are also present in other antimicrobial proteins such as defesins. Hydrophilicity analysis indicated that each of these ABCD loops is markedly hydrophilic. Synthetic peptides, corresponding to these motifs in lactoferrin and lysozyme, exhibited AGE-binding activity. Since diabetes is associated with abnormally high levels of tissue and serum AGEs, the elevated AGEs may inhibit endogenous antibacterial proteins by binding to the conserved ABCD motif, thereby increasing susceptibility to bacterial infections in diabetic individuals. These results may provide a basis for the development of new approaches to prevent diabetic infections.
Allergic airway inflammation encompasses a complex interaction between very heterogeneous cells and mediators of the upper and lower respiratory tract, leading to clinical hallmarks such as allergen-induced airways obstruction, hypersecretion and airway hyperresponsiveness. There is a substantial and growing body of evidence demonstrating striking similarities between allergic diseases of the upper and lower respiratory tract. Recent data indicate that these similarities are not only due to similar patterns of local antigen presentation. Systemic immune mechanisms as well as a complex neuronal dysfunction seem to be crucial to the pathogenesis and outcome of allergic airway inflammation. This article reviews current research on pathophysiology and therapy of this multi-organ disease.
Submucosal glands contribute to airway surface liquid (ASL), a film that protects all airway surfaces. Glandular mucus comprises electrolytes, water, the gel-forming mucin MUC5B, and hundreds of different proteins with diverse protective functions. Gland volume per unit area of mucosal surface correlates positively with impaction rate of inhaled particles. In human main bronchi, the volume of the glands is ∼50 times that of surface goblet cells, but the glands diminish in size and frequency distally. ASL and its trapped particles are removed from the airways by mucociliary transport. Airway glands have a tubuloacinar structure, with a single terminal duct, a nonciliated collecting duct, then branching secretory tubules lined with mucous cells and ending in serous acini. They allow for a massive increase in numbers of mucus-producing cells without replacing surface ciliated cells. Active secretion of Cl(-) and HCO3 (-) by serous cells produces most of the fluid of gland secretions. Glands are densely innervated by tonically active, mutually excitatory airway intrinsic neurons. Most gland mucus is secreted constitutively in vivo, with large, transient increases produced by emergency reflex drive from the vagus. Elevations of [cAMP]i and [Ca(2+)]i coordinate electrolyte and macromolecular secretion and probably occur together for baseline activity in vivo, with cholinergic elevation of [Ca(2+)]i being mainly responsive for transient increases in secretion. Altered submucosal gland function contributes to the pathology of all obstructive diseases, but is an early stage of pathogenesis only in cystic fibrosis.
Copyright © 2015 the American Physiological Society.
Despite the fact that pathogenic infections are widely treated by antibiotics in the clinic nowadays, the increasing risk of multidrug-resistance associated with abuse of antibiotics is becoming a major concern in global public health. The increased death toll caused by pathogenic bacterial infection calls for effective antibiotic alternatives. Lysozyme-coated mesoporous silica nanoparticles (MSNs⊂Lys) are reported as antibacterial agents that exhibit effi cient antibacterial activity both in vitro and in vivo with low cytotoxicity and negligible hemolytic side effect. The Lys corona provides multivalent interaction between MSNs⊂Lys and bacterial walls and consequently raises the local concentration of Lys on the surface of cell walls, which promotes hydrolysis of peptidoglycans and increases membrane-perturbation abilities. The minimal inhibition concentration (MIC) of MSNs⊂Lys is fi vefold lower than that of free Lys in vitro. The antibacterial effi cacy of MSNs⊂Lys is evaluated in vivo by using an intestine-infected mouse model. Experimental results indicate that the number of bacteria surviving in the colon is three orders of magnitude lower than in the untreated group. These natural antibacterial enzyme-modifi ed nanoparticles open up a new avenue for design and synthesis of next-generation antibacterial agents as alternatives to antibiotics.
Nasal mucus is the first-line defense barrier against (aero-) allergens. However, its proteome and function have not been clearly investigated.
The role of nasal mucus in the pathophysiology of allergic rhinitis was investigated by analyzing its proteome in patients with allergic rhinitis (n = 29) and healthy control subjects (n = 29).
Nasal mucus was collected with a suction device, tryptically digested, and analyzed by using liquid chromatography-tandem mass spectrometry. Proteins were identified by searching the SwissProt database and annotated by collecting gene ontology data from databases and existing literature. Gene enrichment analysis was performed by using Cytoscape/BINGO software tools. Proteins were quantified with spectral counting, and selected proteins were confirmed by means of Western blotting.
In total, 267 proteins were identified, with 20 (7.5%) found exclusively in patients with allergic rhinitis and 25 (9.5%) found exclusively in healthy control subjects. Five proteins were found to be significantly upregulated in patients with allergic rhinitis (apolipoprotein A-2 [APOA2], 9.7-fold; α2-macroglobulin [A2M], 4.5-fold; apolipoprotein A-1 [APOA1], 3.2-fold; α1-antitrypsin [SERPINA1], 2.5-fold; and complement C3 [C3], 2.3-fold) and 5 were found to be downregulated (antileukoproteinase [SLPI], 0.6-fold; WAP 4-disulfide core domain protein [WFDC2], 0.5-fold; haptoglobin [HP], 0.7-fold; IgJ chain [IGJ], 0.7-fold; and Ig hc V-III region BRO, 0.8-fold) compared with levels seen in healthy control subjects.
The allergic rhinitis mucus proteome shows an enhanced immune response in which apolipoproteins might play an important role. Furthermore, an imbalance between cysteine proteases and antiproteases could be seen, which negatively affects epithelial integrity on exposure to pollen protease activity. This reflects the important role of mucus as the first-line defense barrier against allergens.
Air humidification is sometimes used to reduce dryness symptoms in temperate climates, but the biological mechanism behind this effect is not well understood. We have investigated the effect of air humidification on physiological signs from the eyes and nose, other medi cal symptoms and on perceived air quality. An experi mental study was performed in two units of a well-venti lated geriatric hospital in southern Sweden. Blinded cen tral steam air humidification in one of the units during 6 weeks raised the relative air humidity to 43% RH (Janu ary-March 1997), while the other unit with 35% RH served as control. All staff (N = 32) working the day shift were invited to undergo a medical investigation per formed in the hospital units. It included measurement of tear film stability, acoustic rhinometry, nasal lavage and completing a medical questionnaire. The investigation was repeated at both units after 6 weeks and 26 subjects participated on both occasions (81 %). The technical mea surements were room temperature, relative air humidity
Interleukin (IL)-4 causes the dose limiting sensation of nasal congestion when administered systemically at doses of 3 mug/kg or higher thrice daily to humans. This side effect was observed in a group of patients treated as part of an immunotherapy protocol for cancer management. To determine the source of this congestion, nasal secretions were collected prospectively in a group of patients at baseline and after provocation with normal saline, methacholine (which stimulates glandular secretion), and histamine (which causes increased vascular permeability). Nasal lavages obtained at baseline and after provocation were analyzed for the presence of these glandular and vascular proteins and inflammatory mediators. Washings and provocations were performed before IL-4 administration, after 24 hours of IL-4 treatment, and after 3 days of treatment, at a time when nasal congestion was maximal. Compared with histamine challenge before IL-4 treatment, the secretion of the plasma proteins albumin and IgG were significantly decreased after 3 days of IL-4 treatment. IL-4 treatment had no apparent effect on methacholine-induced responses. Thus systemically administered IL-4 causes the subjective sensation of nasal congestion, increased histamine in nasal lavages, and the development of vascular unresponsiveness to histamine, without affecting parasympathetic responses to histamine. The relationships among increases in nasal lavage histamine, vascular unresponsiveness to histamine, and the sensation of nasal congestion are unclear.
The upper respiratory tract, extending from the nares to the larynx, performs several essential physiological functions, including olfaction, phonation, air conditioning, filtering, and microbial defense. In the occupational setting, workers are exposed to a wide variety of chemical and biological agents that can have allergic or irritant effects, or both, on the upper airway. This article presents our current understanding of the pathophysiology, clinical assessment, and management of occupationally related upper airway disorders.
Human parotid and submandibular glands were studied by paired immunofluorescence staining, including a variety of combinations of fluorochrome conjugates with contrasting colors. Lactoferrin (Lf), secretory component (SC), and particularly amylase were demonstrated in serous acinar cells of both glands. In addition, lysozyme (Ly) was present in some acini, although mainly located in intercalated ducts where Lf was also most commonly seen. SC was present in acini, intercalated ducts, and striated ducts but not in large collecting ducts. Staining for SC was generally faint but increased in intensity at the cell periphery and particularly at the luminal face of striated duct cells. Immunoglobulin (Ig) A--and IgM when detectable--showed an epithelial distribution similar to that of SC, in accordance with the known secretory properties of these two Ig classes. Conversely, IgG was not present in epithelial cells, despite its high extravascular concentrations. Mucous epithelial elements did not show unequivocal staining for any of the proteins studied. Formaldehyde fixation, combined with pronase treatment of tissue sections and prolonged exposure (20 hr) to antibody, enhanced markedly the staining intensity for lysozyme; ethanol fixation and 30-min incubation with conjugates generally afforded better localization of the other epithelial components.
The two basic proteins lysozyme and lactoferrin have been isolated from solubilized mucoid sputum from patients with chronic bronchitis in one step by cation exchange chromatography. In sputa from 13 patients with chronic bronchitis their mean concentrations were 0.4 g/l and 0.7 g/l, respectively, representing 6.6% and 11.5% of the total amount of solubilized protein. Lysozyme and the acid mucin glycoproteins of sputum formed aggregates at low ionic strength, probably as a result of electrostatic interactions between the two. Although only aggregates were formed and not a viscoelastic fluid or a gel, these interactions may contribute to the viscoelastic properties of native sputum.
Using commercially available reagents we developed an enzyme-linked immunosorbent assay to measure lactoferrin in normal human tears. Tears obtained from 38 normal human subjects contained a mean lactoferrin content of 2.2 mg/ml (g/l). No differences in the mean level of lactoferrin were found when tears were collected by either Schirmer tear strips or by glass capillaries after short tear gas stimulation. Tear lactoferrin levels were not influenced by age or sex of the subjects investigated in this study. We found that lactoferrin represents approximately 25% by weight of the total tear proteins.
The distribution of lactoferrin and glycoprotein in human bronchial glands has been studied by electron microscopy using an immunoperoxidase method to stain the former and a periodic acid-chromic acid-silver methenamine sequence for the latter, each applied to ultrathin Epon sections. The distribution of lactoferrin corresponds to that of lysozyme. Lactoferrin and lysozyme are both confined to serous acini where the granules show a variable pattern of staining. Some serous granules are filled uniformly with lactoferrin, some lack lactoferrin in a small central core or a thin peripheral rim, and some are completely devoid of lactoferrin. Glycoprotein is present in all mucous granules but only certain serous granules. The latter may be filled uniformly with glycoprotein or glycoprotein may form a thin peripheral coat about centrally located lactoferrin and lysozyme. An electron-dense central core found in some serous granules contains neither glycoprotein, lactoferrin, nor lysozyme.
The lactoferrin content of human plasma has been measured by an enzyme-linked immunosorbent assay. In cord blood the level was 0.02-0.3 mg/l, corresponding to 3–44 × 10-10 mol/l lactoferrin; in plasma 5 d post partum the level had not changed. In adults the level was 0.02-0.2 in 29 out of 30 plasma samples and above 1 mg/l in 1 sample. Similar results were obtained with EDTA, citrate or heparin as anti-coagulant.
The iron-binding protein characteristic of milk, here called lactoferrin, has been demonstrated to occur in saliva, nasal secretions, tears, bronchial mucus, hepatic bile, pancreatic juice, seminal fluid, female cervical mucus and urines.It is suggested that its iron-binding properties are of value in the defence of epithelial surfaces against infection.
Baseline rates for secretion of mucous glycoprotein were similar similar (680--830 microgram/g tissue/24 hour) for cultured tracheal epithelium from newborns of 26--32 weeks' gestation, full term newborns, and older children. Addition of methacholine to culture medium augmented secretory rates of glycoprotein from all tissue sources 3--5 fold. The overall composition of secreted mucous glycoproteins changed little with increasing age. A trend toward less sulfation and toward increased sialic acid and fucose content was noted in secreted glycoproteins from explants of older subjects. Histochemical observations of stored glycoprotein in tracheal tissue, which was subsequently used for organ culture experiments, confirmed that a modest, but consistent sulfate to sialic acid shift occurs during early life. In contrast, baseline secretory rates for lysozyme from tracheal epithelium of preterm infants were one-half as large as rates from epithelium of full term babies and were refractory to cholinergic stimulation. Stimulation of lysozyme secretion by a cholinergic agonist was achieved in all cases by 40 weeks' gestation. We conclude that basal glycoprotein secretion and the mechanism for glycoprotein response to cholinergic stimulation have developed by the earliest age of viability, but that lysozyme secretion is deficient and is unresponsive to cholinergic stimulation in tracheal tissue from preterm newborns.
The distribution of lysozyme (LZM) in normal human tissues was determined with the use of the immunoglobulin-enzyme (peroxidase) bridge method. LZM was detected in the following cells and tissues: secretory cells of the lacrimal gland, ductal epithelial cells of the parotid gland and the serous parts of the mixed sublingual glands, the esophageal submucosal glands, bronchial serous submucosal glands, gastric and pyloric glands, Brunner's glands of the duodenum, the Paneth cells of the small intestine, Kupffer cells of the liver and renal proximal tubular cells. In addition, LZM was also found in the mononuclear or polymorphonuclear cells of the placenta, lung, lamina propria of the small intestine, lymph nodes and spleen. This distribution of LZM is discussed in relation to its possible physiologic role in human tissues and particularly to its known antibacterial properties.
Monoclonal antibodies against human lactoferrin define at least 3 and possibly as many as 6 different epitopes. A sandwich enzyme-linked immunoassay, using monoclonals against different epitopes, has been optimised for the measurement of serum lactoferrin. In 35 samples from healthy adults the mean lactoferrin content of serum from blood clotted overnight was 0.54 +/- 0.26 micrograms/ml.
Histamine release occurs during the late phase allergic reaction concomitantly with neutrophil (PMN) infiltration. To determine whether PMN might release a factor capable of causing histamine release, supernatants generated by incubating human PMN in the presence or absence of specific activators were added to rat basophilic leukemia cells (RBL) and histamine release was measured. PMN supernatants from 17 of 21 donors induced noncytotoxic histamine release. Neutrophil-derived histamine-releasing activity, termed HRA-N, was dose-dependent and supernatants from greater than or equal to 10(7) PMN/ml caused 6 to 27% net histamine release from RBL. PMN supernatants induced histamine release as effectively as did intact PMN cocultured with RBL. The capacity of various donors to generate HRA-N was not related to atopic status or gender but was inversely related to the proportion of eosinophils (EOS) contaminating the PMN isolate (the larger the proportion of EOS, the lower the histamine release). Addition of EOS to PMN during the generation of HRA-N completely inhibited histamine-releasing activity. HRA-N was not released from mononuclear cells or platelets contaminating the PMN preparation. HRA-N release was not increased by the presence of either serum-treated zymosan or phorbol myristate acetate, agents that caused dose-related release of PMN granule enzymes. Indeed, HRA-N was released from unstimulated PMN in the complete absence of granule enzyme release. HRA-N release was detectable by 15 min and the majority of release occurred between 45 and 60 min of incubation. Thus, the data indicate that HRA-N is released spontaneously from human PMN and that HRA-N release is independent of primary or secondary PMN granule release. It is attractive to suggest that release of HRA-N by PMN might act to recruit mast cells or basophils into participating in acute inflammatory reactions.
The consumption of certain foods causes watery rhinorrhea (gustatory rhinitis) in many individuals. To examine the underlying mechanisms responsible for this common phenomenon, 12 subjects ingested control foods and positive foods (foods that cause rhinorrhea). Nasal lavages performed 10 minutes after each food challenge were analyzed for albumin and total protein. Positive food challenge, but not control food challenge, induced rhinorrhea in all subjects. Positive food challenge increased albumin (7.8 +/- 1.9 to 24.5 +/- 7.6 mg/L; p less than 0.025) and total protein (79 +/- 9 to 258 +/- 41 mg/L; p less than 0.001) without altering the ratio of albumin to total protein (albumin percent). Nasal pretreatment with atropine clinically blocked the positive food-induced rhinorrhea and significantly inhibited secretion of both albumin and total protein, again without affecting the albumin percent. Thus, gustatory rhinitis is produced by spicy foods that stimulate atropine-inhibitable muscarinic receptors (probably on submucosal glands), and the syndrome can be treated prophylactically by use of topical atropine.
Nasal provocation tests were performed on nine atopic and 15 nonatopic subjects in order to assess the sources of protein in histamine-induced secretions and to examine the bilateral secretory response to unilaterally applied topical histamine (the nasonasal reflex). Nasal lavages were assayed for the following proteins: albumin, total protein, secretory IgA (sIgA), nonsecretory IgA (serum IgA), and total IgA. Histamine stimulation produced a profound ipsilateral protein secretion enriched in the serum proteins albumin and nonsecretory IgA. Histamine also produced a smaller contralateral protein secretion (about 15% as large as the ipsilateral response) which contained disproportionately elevated concentrations of the glandular protein sIgA. Topical pretreatment with chlorpheniramine (an H-1 antihistamine) completely abrogated the ipsilateral nasal secretory response to histamine. Nasal pretreatment with atropine (a muscarinic antagonist) had no significant effect on ipsilateral nasal secretion and did not alter the capacity of histamine to stimulate contralateral secretions (the nasonasal reflex). Histamine therefore stimulates secretion by both a direct action that increases plasma protein extravasation and by an indirect reflex mechanism that stimulates glandular secretion.
To determine whether biologically active products of eosinophils, neutrophils and complement contribute to the development of adult respiratory distress system (ARDS) we measured eosinophil cationic protein (ECP), lactoferrin (LF) and C3a in bronchoalveolar lavage (BAL) and blood by means of radioimmunoassays. Seventeen patients served as controls. Fifteen patients were studied before and after major surgery to evaluate the influence of the surgical procedure, and 12 patients with ARDS were investigated 4-12 h after the onset of the disease. Major surgery per se significantly increased ECP in BAL, LF in serum and C3a in BAL and plasma. ECP, LF and C3a levels in BAL and blood were all significantly higher in ARDS patients as compared with levels in controls and those observed after major surgery. The higher ECP levels in BAL were associated with the more severe ARDS as was also the case for C3a in BAL and plasma and LF in serum. One out of 15 patients subjected to major surgery developed ARDS postoperatively and had very high levels of ECP, LF and C3a in BAL and blood at sampling 3 h prior to onset of ARDS, and these levels were similar to those observed in ARDS patients. One out of 12 ARDS patients died from the disease and this patient had the highest level of ECP in BAL and serum. Our results strongly support the role of activated polymorphonuclears, and notably the activated eosinophils, in the pathogenesis of ARDS. Evidence is also presented that ECP can be used as a predictor of impending ARDS.
Lactoferrin, an iron complexing protein in normal tears, is an important component of the nonspecific host defense system of the external eye. We measured tear lactoferrin levels in patients with contact lens-induced giant papillary conjunctivitis (GPC) by an enzyme-linked immunosorbent assay (ELISA). Patients with active GPC (N = 26) had significantly reduced tear levels of lactoferrin (0.876 +/- 0.42 mg/ml) compared with normal individuals (N = 12; 1.73 +/- 0.46 mg/ml, P less than 0.0003) and the control contact lens wearers' group (N = 11; 1.57 +/- 0.92 mg/ml, P less than 0.003). Patients with vernal conjunctivitis (N = 10), an ocular disease with similar histopathology, had slightly reduced concentrations of tear lactoferrin (1.22 +/- 0.59 mg/ml). Patients with inactive GPC (N = 7) had normal tear levels of lactoferrin (1.33 +/- 0.49 mg/ml). The lactoferrin to total protein ratio in the tears was significantly reduced in patients with GPC compared to normal subjects, control contact lens wearers, and patients with inactive GPC. The decreased tear levels of lactoferrin in patients with GPC may contribute to increased coating of lenses with bacteria and their products and enhanced ocular inflammation which may play a role in the pathogenesis of GPC.
Nasal provocation tests with normal saline and methacholine (MC) were performed in 25 atopic and 27 nonatopic subjects in an effort to assess the sources of protein in induced airway secretions. Nasal lavages obtained at baseline and after provocation were analyzed for albumin, total protein, secretory IgA (sIgA), and total IgA. Compared with baseline levels or saline provocation, MC provocation increased the secretion of albumin (p less than 0.025), total protein (p less than 0.001), sIgA (p less than 0.025), and total IgA (p less than 0.025), but did not significantly affect the relative proportions of albumin-to-total protein (albumin percent) or sIgA-to-total IgA (sIgA/total IgA ratio). Nasal pretreatment with atropine significantly inhibited MC-induced secretion of all 4 proteins, again without affecting the albumin percent or the sIgA/total IgA ratio. Because MC is known to stimulate atropine-inhibitable secretion of glandular products, these data suggest that sIgA and albumin may accompany glandular secretions. Immunohistochemical analyses of nasal turbinates confirmed that secretory component was found only on serous cells within submucous glands. Thus, it appears that cholinergic stimulation may regulate sIgA secretion and thereby participate in local nasal (and possibly respiratory tract) immunity.
Normally the daily volume of lower respiratory tract secretions, in man, is probably less than 100 ml. In hypersecretory disease the volume increases sufficiently to cause cough and expectoration of secretions as sputum. The proportions which are sol or gel vary in disease as does the way in which constituent molecules partition in each phase. The constituent molecules and the cells which produce them (indicated in parentheses) may be classified as follows: 1. Mucus-glycoproteins present as droplets, or sheets (produced by mucous cells), periciliary fluid (serous or ciliated cell or a transudate), surface muco-substance (all epithelial cells) or surfactant hypophase (Clara or type II alveolar cells). 2. Proteins and peptides such as lysozyme (serous cell and macrophage), lactoferrin (serous cell and neutrophil), secretory piece (surface epithelium and submucosal glands), regulatory neuropeptides (dense-core granulated cell and both motor and sensory nerves) and fibronectin (alveolar macrophages). 3. Glycosaminoglycans such as heparan sulphate (epithelial membranes), heparin (mast cell), chondroitin sulphates and hyaluronate (connective tissue constituents). 4. Lipids including triglycerides (stored in cells) glycolipids (cell membrane), phospholipids (type II alveolar cells), sphingolipids (cell membrane), steroids (? Clara cells) and terpenes (cell membrane). 5. Anti-proteases and anti-oxidants such as bronchial protease inhibitors (serous anc Clara cells), alpha-2-macroglobulin (macrophage), alpha-1-antitrypsin (transudate) and anti-oxidants (type II alveolar cell and macrophage). 6. Other 'secretions' including ions and water (surface epithelium and submucosal glands), mediators of inflammation (migratory cell granules and their membranes), and serum proteins (present in transudate/exudate).
The distribution of lysozyme in normal gastric and duodenal mucosa was studied by light- and electron-microscopic immunocytochemical techniques (direct enzyme-labeled antibody method). In the duodenal mucosa, lysozyme was found in the Paneth cells and the epithelial cells of Brunner's glands. Electron-microscopically, lysozyme was found in rough endoplasmic reticulum and perinuclear spaces, which were assumed to be protein-synthesizing organelles, and also in the secretory granules of Paneth cells. Additionally, lysozyme was detected in the stomach in mucinous granules and in some parts of the rough endoplasmic reticulum within the epithelial cells of the pyloric glands, the mucous neck cells of the fundic glands, and in several surface epithelial cells of the plyoric and fundic regions. This suggests that some quantity of lysozyme in gastrointestinal secretion originates from the gastric and duodenal glands, and that it acts as a defense mechanism in the gastrointestinal tract.
A non-competitive avidin-biotin immuno-enzymatic assay (NABA) for lysozyme is described. The assay was found to be more sensitive than a competitive assay with biotinylated lysozyme. The lower detection limit of NABA was 0.1 ng lysozyme/ml compared to 1 ng/ml for the competitive assay. The intra-assay and inter-assay coefficients of variation for NABA were 5.9 and 9.1%, respectively. The total time for NABA can be decreased (to less than 1 h) without influencing the detection limit or the analytical range. Serum lysozyme levels measured by NABA and the enzymatic assay in 32 samples showed a correlation coefficient of r = 0.97.
Serum and salivary total IgA, IgG and IgM as well as salivary innate non-immunoglobulin antimicrobial factors (lysozyme, lactoferrin, salivary and leukocyte peroxidase systems) were measured in 13 children prone to recurrent respiratory infections and compared to their age-matched healthy controls. Serum IgG and IgM levels were significantly elevated and salivary IgA remarkably low in infection-prone children as compared to the controls. However, the levels of secretory piece-bearing IgA were about the same in both groups. There were no significant differences between the two groups in serum IgA levels or in any of the non-immunoglobulin factors. The results indicate that low salivary IgA is associated with recurrent respiratory infections.
The concentrations of serum albumin (SA) in the conjunctival fluid were measured in 25 patients before surgery and in the post-operative period following cataract extraction. We found a significant increase in mean tear fluid SA concentration on the first post-operative day. The SA concentration remained high during the first 3 pre-operative levels. No correlation between the post-operative concentration profile of tear fluid SA and earlier findings of the tear lactoferrin (LF) profile of the same group of patients (Jensen et al. 1985) could be shown, although a trend towards an inverse relationship was apparent from the graphs. Pre-operatively, we found a significant positive correlation between age and concentration of SA in the conjunctival fluid (P less than 0.05) which might be interpreted as an increase in leakage from conjunctival vessels with age. This would, at least partly, explain the decreasing LF concentration with age (Jensen et al. 1986; McGill et al. 1984). It is concluded that transudate/exudate from the conjunctival vessels, represented by the change in the SA concentration in the conjunctival fluid, might be responsible for an initial post-operatively reduced concentration of LF and possibly other lacrimal gland proteins.
Previously described methods for measuring human tear lysozyme are fraught with shortcomings. A new method has been devised. Tear fluid was collected on Whatman filter paper discs. Each disc was placed in a tightly capped tube containing sodium phosphate buffer. Fluid from each tube was placed directly into a well of the lysozyme immunodiffusion plate. After the precipitation rings had reached maximum size, their diameters were measured. A linear standard curve was constructed, and lysozyme concentration was expressed as micrograms per milliliter. The tear lysozyme concentration was obtained from the standard curve and corrected for the assay dilution factor. The mean tear lysozyme concentration in 15 normal patients was 1.4 +/- 0.5 mg/mL. In ten patients with dry eyes, the mean was 0.7 +/- 0.5 mg/mL. The method used to collect, store, and transport tears is easily performed in the clinic and readily tolerated by patients. The technique of radial immunodiffusion is reliable and simple, compared with other assays.
Recent studies suggest that salivary lysozyme (Lz), lactoferrin (Lf), peroxidase (Spx), and secretory immunoglobulin A (sIgA) may interact in a common antimicrobial system. A multiple protein approach therefore may be needed to determine the role of this system in oral health and ecology. In the present study we investigate the relationships between levels of Lz, Lf, Spx, and sIgA (adjusted for flow rate and total protein) in stimulated parotid saliva from 44 dental students. Principal components analysis was used to determine major patterns of intercorrelation between variables; cluster analysis was used to identify groups of subjects with similar salivary profiles for Lz, Lf, Spx, and sIgA. Spx tended to vary independently of Lz and Lf, which, in turn, tended to vary together. sIgA showed a weak negative relationship with Spx and a weak positive relationship with Lz and Lf. Six major clusters of subjects with similar antimicrobial protein profiles were found. These were significantly different at P less than 0.0001. Spx was the most important determinant of cluster membership followed (in order of importance) by Lz, Lf, and sIgA. Cluster profiles were Spx-, sIgAmu, Lf-, Lz-; Spx-, sIgA+, Lfmu, Lz+; Spxmu, sIgAmu, Lfmu, Lzmu; Spx+, sIgA-, Lf-, Lz-; Spx+, sIgAmu, Lf+, Lz-; and Spx+, sIgAmu, Lf+, Lz+ (-, mu, and + refer to the position of the cluster mean each variable relative to the overall mean for that variable). Results suggest that clusters may be a product of independent variation in the secretory activity of acinar and intercalated duct cells.
A sensitive and specific enzyme linked immunoassay for lactoferrin is described. The assay is designed to estimate lactoferrin concentrations in samples of duodenal aspirate, but is suitable for other biological fluids as well. The detection limit was 4.7 pmol/l. Within and between assay reproducibility was 41 and 178 pmol/l (1 SD), respectively, at a concentration of 1974 pmol/l. In the fasting state the concentration of lactoferrin in duodenal aspirates of three normal subjects ranged from 0-390 pmol/l and increased to 2860-14,820 pmol/l after pancreatic stimulation.
Lysozyme is a bacteriolytic enzyme found in respiratory tract fluid. In this study, immunocytochemistry was used to determine the cells of origin of tracheal lysozyme in the ferret. Lysozyme was found in secretory granules of serous but not mucous cells in the submucosal glands, and was absent from the surface epithelium, cartilage, and connective tissue. The exclusive presence of lysozyme in serous gland cells renders it useful as a biochemical marker of that cell type.
Measurements of lysozyme assayed from the incubating medium indicated that bethanechol stimulated lysozyme release by 260±80.9% (mean ±SE), phenylephrine by 80±16.4%, and terbutaline by 25±10.2%. Electron-microscopic and immunocytochemical analysis of incubated tissues revealed loss of serous granules and lysozyme immuno-reactivity in response to the drugs. Atropine, propranolol, and phentolamine blocked the stimulatory effects of bethanechol, terbutaline, and phenylephrine, respectively.
These findings establish the usefulness of lysozyme as a serous-cell marker and demonstrate that secretory responses of different magnitude are evoked by equimolar concentrations of alpha- and beta-adrenergic and cholinergic drugs.
An enzyme-linked immunosorbent assay has been developed for quantitation of lactoferrin (LF) in body fluids. An indirect double-sandwich method was used which allows a sensitivity of 3 ng LF/ml in samples of polymorphonuclear cell lysates and serum. Mean LF content of serum was 0.307 +/- 0.066 micrograms/ml (n = 18). Mean LF content of polymorphonuclear cells was 4.90 +/- 1.48 micrograms/10(6) PMN. Concentrations of LF were similar in serum and in plasma of EDTA anticoagulated blood. Advantages of this method include its rapidity, and radioactivity is not required.
Concentrations of lysozyme, lactoferrin, ceruloplasmin, IgA, and IgG have been measured in tears by the ELISA (enzyme-linked immunosorbent assay) technique. Tears were collected on weighed filter paper discs, after which they were eluted into buffer and transported frozen to a remote laboratory for assay. Patients with sicca, questionably dry eyes and ocular pemphigoid were sampled, as were 54 normal volunteers. Tear protein profiles were established which were unique for each condition and clearly differed from the normal controls. The assay developed is considered suitable for other proteins such as IgE, and could also be used for monitoring the effects of drugs on the lacrimal gland.
In tear fluid, a large number of proteins can be detected with electrophoretic or immunologic techniques. The composition of serum proteins in tears resembles that of whole serum. By comparison of the protein patterns resulting from different sampling methods, it was shown that serum albumin is not present in the secretion of the lacrimal gland, but is mixed with the tear fluid in the conjunctival sac. The in vitro synthesis and excretion of more than 20 protein components by the lacrimal gland could be demonstrated. Among these were lactoferrin, tear-specific prealbumin, lysozyme, and secretory IgA. The complexity of the electrophoretic protein pattern of tear fluid can be explained from the combination of the secretory activity of the lacrimal gland and the leakage of serum proteins from the circulation into the tear fluid.
The lactoferrin content of human plasma has been measured by an enzyme-linked immunosorbent assay. In cord blood the level was 0.02-0.3 mg/l, corresponding to 3-44 X 10-(10) mol/l lactoferrin; in plasma 5 d post partum the level had not changed. In adults the level was 0.02-0.2 in 29 out of 30 plasma samples and above 1 mg/l in 1 sample. Similar results were obtained with EDTA, citrate or heparin as anti-coagulant.
Unstimulated basal tears and stimulated tears were collected from normal controls, keratoconjunctivitis sicca (KCS) patients, and contact lens (CL) wearers. Basal tear volume (Periopaper) and reflex tear volume (Schirmer Strip) were measured and tears eluted from their respective strip with a TMED-acetic acid buffer. Lactoferrin and lysozyme concentrations were measured in the eluates. Concentration of lysozyme in basal tears was: 65 +/- 43 mg% in normals; 44 +/- 26 mg% in KCS, and 81 +/- 50 mg% in CL wearers. Concentration of lysozyme in reflex tears was: 160 +/- 73 mg% in normals, 74 +/- 41 mg% in KCS, and 186 +/- 83 mg% in CL wearers. Concentration of lactoferrin in basal tears was 137 +/- 102 mg% in normals, 154 +/- 82 mg% in KCS, and 157 +/- 80 mg% in CL wearers. Concentrations of lactoferrin in reflex tears was: 327 +/- 187 mg% in normals; 171 +/- 69 mg% in KCS, and 302 +/- 160 mg% in CL wearers. The lactoferrin concentration in all tear samples was consistently higher (1.6-3.5 times) than lysozyme. In basal tears, lactoferrin concentrations were not significantly different in the three groups. In reflex tears, however, lactoferrin was significantly lower in KCS than normal or CL wearers. The concentration of lysozyme in both basal and reflex tears was significantly lower in the KCS when compared to normal or CL wearers. Lysozyme and lactoferrin concentrations in both basal and reflex tears were similar in CL wearers and normal controls. The sampling and elution of basal and reflex tears as described appears to be a feasible technique for analysis of lysozyme and lactoferrin concentrations by the electroimmunodiffusion technique and has potential diagnostic value.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Reliability of human tear protein identification assay is improved by HPLC fractionation of tear samples
- R J Fullard
Fullard, R. J. 1988. Reliability of human tear protein identification assay is improved by HPLC fractionation of tear samples. Invest. Ophthalmol. & Visual Sci. 29(Suppl):47.
In Laboratory Manual of Neutrophil Function
- Lysozyme
Lysozyme. In Laboratory Manual of Neutrophil Function. Raven
Press, New York. 149-150.
The origins of secretions in the lower respiratory tract Relationship between levels of lysozyme, lactoferrin, salivary peroxidase, and secretory immunoglobulin A in stimulated parotid saliva
- P K Jeffrey
Jeffrey, P. K. 1987. The origins of secretions in the lower respiratory tract. Eur. J. Respir. Dis. 7 1(Suppl 153):34-42.
31. Rudney, J. D., and Q. T. Smith. 1985. Relationship between
levels of lysozyme, lactoferrin, salivary peroxidase, and secretory immunoglobulin A in stimulated parotid saliva. Infect. Immun. 49:469-
475.
A monoclonal antibody-based immunoassay for human lactoferrin An enzyme-linked immunoassay (ELISA) for measurement oflactoferrin
- S V Hetherington
- J K Spitznagel
- P G Quie
A monoclonal antibody-based immunoassay for human lactoferrin. J. Immunol. Methods. 84:135-141.
18. Hetherington, S. V., J. K. Spitznagel, and P. G. Quie. 1983. An
enzyme-linked immunoassay (ELISA) for measurement oflactoferrin.