Chemokines and cytokines may occur in dentinal fluids in response to local infection and inflammation. To test this hypothesis, we assessed the presence and concentration of inflammatory mediators in fluid extracted from the coronal occlusal dentin of trimmed teeth.
Freshly extracted sound, carious, and restored molars were trimmed through the enamel to expose the underlying dentin, etched with 35% phosphoric acid, and rinsed. Fluid was extracted from the coronal occlusal dentin of these trimmed teeth by centrifugation at 2,750 × g for 30 minutes.
When assessed by MALDI-TOF, fluid extracted from the coronal occlusal dentin from 16 molars contained at least 117 peaks with different masses suggesting that this fluid was rich with molecules within the appropriate mass range of potential mediators. Indeed, when assessed for chemokines and cytokines, fluid extracted from the coronal occlusal dentin from 25 extracted molars with caries lesions, 10 extracted restored molars with occlusal amalgam, and 77 extracted sound molars contained IL-1β, TNF-α, IL-6, IL-8, IL-12(p70), and IL-10. A significant elevation was found for TNF-α (p=0.041) in extracted fluid from teeth restored with amalgam fillings.
Overall, fluid extracted from the coronal occlusal dentin of trimmed teeth may be useful in identifying proteins and other molecules in dentin and pulpal fluids and determining their role as mediators in the pathogenesis of oral infection and inflammation.
Dentinal fluid; Chemokines; Cytokines; MALDI-TOF
Human β defensin DEFB103 acts as both a stimulant and an attenuator of chemokine and cytokine responses: a dichotomy that is not entirely understood. Our predicted results using an in silico simulation model of dendritic cells and our observed results in human myeloid dendritic cells, show that DEFB103 significantly (p < 0.05) enhanced 6 responses, attenuated 7 responses, and both enhanced/attenuated the CXCL1 and TNF responses to Porphyromonas gingivalis hemagglutinin B (HagB). In murine JAWSII dendritic cells, DEFB103 significantly attenuated, yet rarely enhanced, the Cxcl2, Il6, and Csf3 responses to HagB; and in C57/BL6 mice, DEFB103 significantly enhanced, yet rarely attenuated, the Cxcl1, Csf1, and Csf3 responses. Thus, DEFB103 influences pro-inflammatory activities with the concentration of DEFB103 and order of timing of DEFB103 exposure to dendritic cells, with respect to microbial antigen exposure to cells, being paramount in orchestrating the onset, magnitude, and composition of the chemokine and cytokine response.
The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9 mg/ml; SPLUNC1 concentrations ranged from 34.7 ng/ml to 13.8 μg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7 ng/ml to 5.3 μg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies.
SPLUNC; SPLUNC1; Luminex; xMAP; saliva; innate immunity
The concept of antimicrobial peptides (AMPs) as potent pharmaceuticals is firmly established in the literature, and most research articles on this topic conclude by stating that AMPs represent promising therapeutic agents against bacterial and fungal agents. Indeed, early research in this field showed that AMPs were diverse in nature, had high activities with low minimal inhibitory concentrations, had broad spectrums of activity against bacterial, fungal and viral pathogens, and could easily be manipulated to alter their specificities, reduce their cytotoxicities and increase their antimicrobial activities. Unfortunately, commercial development of these peptides, for even the simplest of applications, has been very limited. With some peptides there are obstacles with their manufacture, in vivo efficacy and in vivo retention. More recently, the focus has shifted. Contemporary research now uses a more sophisticated approach to develop AMPs that surmount many of these prior obstacles. AMP mimetics, hybrid AMPs, AMP congeners, cyclotides and stabilised AMPs, AMP conjugates and immobilised AMPs have all emerged with selective or ‘targeted’ antimicrobial activities, improved retention, or unique abilities that allow them to bind to medical or industrial surfaces. These groups of new peptides have creative medical and industrial application potentials to treat antibiotic-resistant bacterial infections and septic shock, to preserve food or to sanitise surfaces both in vitro and in vivo.
Antimicrobial peptide mimotopes; Hybrid antimicrobial peptides; Antimicrobial peptide congeners; Stabilised antimicrobial peptides; Antimicrobial peptide conjugates; Immobilised antimicrobial peptides; Cyclotides
There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity—the sphingoid bases d-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid—against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P < 0.0001) for each bacterial species except Serratia marcescens and Pseudomonas aeruginosa. d-Sphingosine (MBC range, 0.3 to 19.6 μg/ml), dihydrosphingosine (MBC range, 0.6 to 39.1 μg/ml), and phytosphingosine (MBC range, 3.3 to 62.5 μg/ml) were active against all bacteria except S. marcescens and P. aeruginosa (MBC > 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection.
Antimicrobial peptides (AMPs) are among the repertoire of host innate immune defenses. In the oral cavity, several AMPs are present in saliva and have antimicrobial activities against oral bacteria, including Streptococcus mutans, a primary etiologic agent of dental caries. In this study, we hypothesized that unique S. mutans strains as determined by DNA fingerprinting from sixty 13 year-old subjects with or without caries experience would have different susceptibilities to α-defensins-1-3 (HNP-1-3), β-defensins-2-3 (HBD-2-3) and LL-37. The salivary levels of these peptides in subjects also were measured by enzyme-linked immunosorbent assays (ELISA). We found that S. mutans strains from caries-active subjects showed greater resistance to salivary HNP-1-2, HBD-2-3 and LL-37 at varying concentrations than those from caries-free subjects. In addition, combinations of these peptides increased their antimicrobial activity against S. mutans either additively or synergistically. The salivary levels of these peptides were highly variable among subjects with no correlation to host caries experience. However, the levels of a number of these peptides in saliva appeared to be positively correlated within an individual. Our findings suggest that the relative ability of S. mutans to resist host salivary AMPs may be considered a potential virulence factor for this species such that S. mutans strains that are more resistant to these peptides may have an ecological advantage to preferentially colonize within dental plaque and increase the risk of dental caries.
Dental caries; defensins; S. mutans; innate immunity; saliva
The purpose of this study was to determine the presence and relative composition of neutral lipids in human saliva.
Whole unstimulated saliva was collected from 12 subjects ranging from 21 to 29 years old. Samples were lyophilized, and lipids were extracted using chloroform-methanol. Lipids were analyzed by thin-layer chromatography.
Human saliva contains cholesterol, fatty acids, triglycerides, wax esters, cholesterol esters and squalene. The mean total neutral lipid content was 12.1 +/− 6.3 µg/ml.
This lipids in human saliva closely resemble the lipids found on the skin surface. These salivary lipids are most likely produced by the sebaceous follicles in the oral mucosa and sebaceous glands associated with major salivary glands.
The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects.
Materials and Methods
Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and 2 healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analyzed utilizing a multiplexed immunoassay (Luminex®). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests.
Compared to healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of IL-1α, IL-1β, IL-6, IL-12 (p40) (pro-inflammatory cytokines); IL-8, MCP-1, MIP-1α, RANTES (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 (regulator of T-cells and NK cells). Smokers displayed decreased amounts of pro-inflammatory cytokines (IL-1α, IL-6, IL-12 (p40)), chemokines (IL-8, MCP-1, MIP-1, RANTES) and regulators of T-cells and NK cells (IL-7, IL-15).
Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.
Smoking; periodontal disease; immune response; periodontitis/etiology; chronic periodontitis; gingival crevicular fluid
Antimicrobial peptides coupled to a ligand, receptor or antibody for a specific pathogenic bacteria could be used to develop narrow-spectrum pharmaceuticals with ‘targeted’ antimicrobial activity void of adverse reactions often associated with the use of broad-spectrum antibiotics. To assess the feasibility of this approach, in this study sheep myeloid antimicrobial peptide (SMAP) 28 was linked to affinity- and protein G-purified rabbit immunoglobulin G (IgG) antibodies specific to the outer surface of Porphyromonas gingivalis strain 381. The selective activity of the P. gingivalis IgG–SMAP28 conjugate was then assessed by adding it to an artificially generated microbial community containing P. gingivalis, Aggregatibacter actinomycetemcomitans and Peptostreptococcus micros. The specificity of the P. gingivalis IgG–SMAP28 conjugate in this mixed culture was concentration-dependent. The conjugate at 50 μg protein/mL lacked specificity and killed P. gingivalis, A. actinomycetemcomitans and P. micros. The conjugate at 20 μg protein/mL was more specific and killed P. gingivalis. This is an initial step to develop a selective antimicrobial agent that can eliminate a specific periodontal pathogen, such as P. gingivalis, from patients with periodontal disease without harming the normal commensal flora.
Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans; Peptostreptococcus micros; Cathelicidins; Targeted antimicrobial activity; SMAP28
Antibiotic therapy is often used with mechanical therapy to treat periodontal disease. However, complications associated with antibiotic use can occur. A ‘bacteria-specific’ targeted approach would eliminate some of these complications and kill specific periodontopathogens without harming the commensal bacteria. One such approach is to couple antimicrobial peptides to a ligand, pheromone, or antibody specific for the periodontopathogen, Porphyromonas gingivalis. To assess the feasibility of this approach, we attached PQGPPQ, a peptide from proline-rich protein 1 to either the N-terminus of SMAP28 (peptide ZS37-37) or the C-terminus of SMAP28 (peptide ZS37-38) to see whether it has potential as a carrier ligand to deliver SMAP28 to the surface of P. gingivalis. For Escherichia coli and Aggregatibacter actinomycetemcomitans, the median minimal inhibitory concentration (MIC) of ZS37-37 was higher than the median of SMAP28 alone, although the median MIC of ZS37-38 was lower than that of SMAP28 alone. For P. gingivalis, there was no difference in the median MIC values. For S. aureus, the median MIC was higher for ZS37-37 and ZS37-38 compared to SMAP28 alone, particularly for ZS37-38. For Fusobacterium nucleatum, the median MIC values were equal for ZS37-37 and ZS37-38 and higher than the median MIC for SMAP28 alone. Attaching PQGPPQ to SMAP28 did not greatly increase the antimicrobial activity of ZS37-37 or ZS37-38 for P. gingivalis nor substantially decrease the antimicrobial activity of ZS37-37 or ZS37-38 for the four other microorganisms tested. This is an initial step to develop a selective antimicrobial agent that has ‘targeted’ antimicrobial activity without adverse reactions often associated with the use of broad-spectrum antibiotics.
Porphyromonas gingivalis; SMAP28; Periodontal disease; Targeted antimicrobial activity
Human neutrophil peptide α-defensins and human β-defensins are small, well-characterized peptides with broad antimicrobial activities. In mixtures with microbial antigens, defensins attenuate proinflammatory cytokine responses by dendritic cells in culture, attenuate proinflammatory cytokine responses in the nasal fluids of exposed mice and enhance antibody responses in the serum of vaccinated mice. Although the exact mechanisms are unknown, defensins first start by binding to microbial antigens and adhesins, often attenuating toxic or inflammatory-inducing capacities. Binding is not generic; it appears to be both defensin-specific and antigen-specific with high affinities. Binding of defensins to antigens may, in turn, alter the interaction of antigens with epithelial cells and antigen-presenting cells attenuating the production of proinflammatory cytokines. The binding of defensins to antigens may also facilitate the delivery of bound antigen to antigen-presenting cells in some cases via specific receptors. These interactions enhance the immunogenicity of the bound antigen in an adjuvant-like fashion. Future research will determine the extent to which defensins can suppress early events in inflammation and enhance systemic antibody responses, a very recent and exciting concept that could be exploited to develop therapeutics to prevent or treat a variety of oral mucosal infections, particularly where inflammation plays a role in the pathogenesis of disease and its long-term sequelae.
adjuvant; defensin; innate immunity; mucosal immunity; oral inflammation
Our aim is to assess the ability of human neutrophil peptide α-defensins (HNPs) and human β-defensins (HBDs) to attenuate proinflammatory cytokine responses and enhance antibody responses to recombinant hemagglutinin B (rHagB) or recombinant fimbrillin A (rFimA) from Porphyromonas gingivalis 381 in mice.
Materials & methods
In the first study, C57BL/6 mice were given 10 μg rHagB or rFimA without and with 1 μg HNP1, HNP2, HBD1, HBD2 or HBD3. At 24 h, mice were euthanized and cytokine concentrations were determined in nasal wash fluid (NWF), bronchoalveolar lavage fluids, saliva and serum. In the second study, C57BL/6 mice were given 10 μg rHagB or rFimA without and with 1 μg HNPs or HBDs similarly on days 0, 7 and 14. At 21 days, mice were euthanized and rHagB- and rFimA-specific antibody responses were determined in NWF, bronchoalveolar lavage fluids, saliva and serum.
Mice given rHagB + HNP2, rHagB + HBD1 and rHagB + HBD3 produced significantly lower (p < 0.05) IL-6 responses than mice given rHagB alone. Mice given rHagB + HNP1, rHagB + HNP2, rHagB + HBD1 and rHagB + HBD3 produced significantly lower (p < 0.05) keratinocyte-derived chemokine responses than mice given rHagB alone. Mice given rFimA produced very low levels of IL-6 and only moderate levels of keratinocyte-derived chemokine in NWF that were not attenuated by prior incubation of rFimA with any defensin. Mice given rHagB + HNP1 produced a significantly higher (p < 0.05) serum IgG antibody response than mice given rHagB alone and mice given rFimA + HNP2 produced a higher, but not significant, antibody response.
The ability of HNPs and HBDs to attenuate proinflammatory cytokine responses in murine NWF and enhance IgG antibody responses in serum was dependent upon both the defensin and antigen of P. gingivalis.
defensins; fimbriae A; hemagglutinin B; immunity; Porphyromonas gingivalis
Lung disease causes most of the morbidity and mortality in cystic fibrosis (CF). However, understanding its pathogenesis has been hindered by lack of an animal model with characteristic features of CF. To overcome this problem, we recently generated pigs with targeted CFTR genes. We now report that, within months of birth, CF pigs spontaneously develop hallmark features of CF lung disease including airway inflammation, remodeling, mucus accumulation, and infection. Their lungs contained multiple bacterial species, suggesting an equal opportunity host defense defect. In humans, the temporal and causal relationships between inflammation and infection have remained uncertain. To investigate these processes, we studied newborn pigs. Their lungs showed no inflammation, but were less often sterile than controls. Moreover, after intrapulmonary bacterial challenge, CF pigs failed to eradicate bacteria as effectively as wild-type pigs. These results suggest that impaired bacterial elimination is the pathogenic event that initiates a cascade of inflammation and pathology in CF lungs. Finding that CF pigs have a bacterial host defense defect within hours of birth provides an opportunity to further investigate pathogenesis and to test therapeutic and preventive strategies before secondary consequences develop.
Human neutrophil peptide α-defensins (HNPs) and human β-defensins (HBDs) are small well-characterized peptides with broad antimicrobial activities and a diversity of innate immune functions. Although the interactions of defensins with bacteria and their membranes have been well characterized, the interactions of defensins with bacterial adhesins have not. Here we determine if HNPs and HBDs bind to the immobilized adhesins of Porphyromonas gingivalis strain 381, recombinant hemagglutinin B (rHagB) and recombinant fimbrillin A (rFimA), by surface plasmon resonance spectroscopy. Association of HNPs and HBDs with rHagB or rFimA was dose dependent and defensin specific. HBD3, HNP-2, and HNP-1 bound more readily to immobilized rHagB than HBD2 and HBD1 did. HNP-2, HNP-1, and HBD3 bound more readily to immobilized rFimA than HBD1 and HBD2 did. Binding of defensins to adhesins may serve to prevent microbial adherence to tissues, attenuate proinflammatory cytokine responses, and facilitate delivery of bound antigen to antigen-presenting cells with defensin receptors.
For many envisioned applications of lentivirus vectors as tools in respiratory biology and therapeutic gene delivery, the efficiency of gene transfer must be improved. We previously demonstrated stable, persistent (>11 months) in vivo expression following a single application of a feline immunodeficiency virus (FIV)-based lentivirus vector (GP64-FIV) to murine nasal epithelia. Here we investigate the efficacy of repeated administration of lentivirus vectors to the airways. Using quantitative bioluminescent imaging, we found that consecutive daily dosing achieved a linear increase in gene expression and greatly increased the number of epithelial cells targeted. Surprisingly, reporter gene expression also increased additively following each of seven doses of FIV delivered over consecutive weeks (1 dose/week), without the development of systemic or local neutralizing antibodies. This approach enhanced expression of both reporter and therapeutic transgenes. Transduction efficiency achieved following a single dose of FIV expressing mouse erythropoietin was insufficient to increase hematocrit, whereas seven consecutive daily doses significantly increased hematocrit. These unexpected results contrast strikingly with findings reported for adenovirus vectors. Prolonged gene expression has been observed in vivo following a single dose of virus vector; however, depending on the application, repeated administration of vector may be necessary to achieve stable, therapeutic gene expression.
Almost two decades after identification of the CFTR gene, we lack answers to many questions about the pathogenesis of cystic fibrosis (CF), and it remains a lethal disease. Mice with a disrupted CFTR gene have greatly facilitated CF studies, but they fail to develop the characteristic pancreatic, lung, intestinal, liver, and other CF manifestations. Therefore, we produced pigs with a targeted disruption of both CFTR alleles. These animals exhibited defective chloride transport. They also developed meconium ileus, exocrine pancreatic destruction, and focal biliary cirrhosis, replicating abnormalities seen in newborn patients with CF. This swine model may provide opportunities to address persistent questions about CF pathogenesis and accelerate discovery of treatments and preventions.
Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.
Lung tissue removed from neonatal calves with acute Mannheimia haemolytica pneumonia showed that rapid up-regulation of the basal mRNA expression of tracheal antimicrobial peptide (TAP), NF-κB, and intercellular adhesion molecule 1 occurred after infection; TAP and interleukin 8 expression were highly correlated. This work suggests that the coordinated expression of β-defensin and inflammatory elements occurs during bacterial pneumonia.
Anionic peptides (APs) are small anionic antimicrobial peptides composed of 7 aspartic acid residues and are produced in the lungs of humans, sheep, and cattle. Although expression by epithelial cells of some antimicrobial peptides (e.g., β-defensins) of humans and ruminants is increased in response to acute infection, AP expression is not increased during acute infection, which suggests that the expression of the latter peptide is constitutive. In this study, the degree of AP expression during the progression (acute, subacute, and chronic) of bronchopneumonia was determined. Mannheimia (Pasteurella) haemolytica, a known inducer of bovineβ-defensins, was inoculated intrabronchially with a fiber-optic bronchoscope in nine 3-month-old sheep, and tissues were collected at 1, 15, and 45 days postinoculation (p.i.); nine control animals received pyrogen-free saline by the same procedure and were killed at the same time points. In the acute group (1 day p.i.), the lungs had lesions typical of bronchopneumonia and the distribution and intensity of AP immunoreactivity (AP-IR) were similar to those of previous studies (minimal intensity and distribution of AP-IR in bronchiolar epithelial cells). In the subacute group (15 days p.i.), there was prominent hyperplasia of bronchiolar and alveolar epithelial cells, and the chronic group (45 days p.i.) had yet more pronounced hyperplasia. In the subacute and chronic groups, the intensity and distribution of AP-IR in the cytoplasm of hyperplastic bronchiolar and type II alveolar cells were significantly increased compared to those of saline-inoculated and contralateral (noninoculated) lung lobes. Although AP expression appears constitutive, the constitutive production of AP is higher in hyperplastic, less differentiated cells than in fully differentiated, mature cells of the respiratory airways. The increased intensity and distribution of AP-IR in immature (hyperplastic) epithelial cells may be a mechanism by which production of a noninducible antimicrobial is increased temporarily during lesion progression and repair. This increased production of AP by hyperplastic cells may protect the lung against further infection until new, fully differentiated epithelial cells are capable of expressing their own inducible array of antimicrobial peptides.
Gallinacin-3 and gallopavin-1 (GPV-1) are newly characterized, epithelial β-defensins of the chicken (Gallus gallus) and turkey (Meleagris gallopavo), respectively. In normal chickens, the expression of gallinacin-3 was especially prominent in the tongue, bursa of Fabricius, and trachea. It also occurred in other organs, including the skin, esophagus, air sacs, large intestine, and kidney. Tracheal expression of gallinacin-3 increased significantly after experimental infection of chickens with Haemophilus paragallinarum, whereas its expression in the tongue, esophagus, and bursa of Fabricius was unaffected. The precursor of gallinacin-3 contained a long C-terminal extension not present in the prepropeptide. By comparing the cDNA sequences of gallinacin-3 and GPV-1, we concluded that a 2-nucleotide insertion into the gallinacin-3 gene had induced a frameshift that read through the original stop codon and allowed the chicken propeptide to lengthen. The striking structural resemblance of the precursors of β-defensins to those of crotamines (highly toxic peptides found in rattlesnake venom) supports their homology, even though defensins are specialized to kill microorganisms and crotamines are specialized to kill much larger prey.
β2-Integrins are leukocyte adhesion molecules composed of alpha (CD11a, -b, -c, or -d) and beta (CD18) subunit heterodimers. Genetic CD18 deficiency results in impaired neutrophil egress into tissues that varies between conducting airways and alveoli of the lung. In this study, we investigated whether CD18 deficiency in cattle affects proinflammatory cytokine (PIC) expression in pulmonary tissue after respiratory infection with Pasteurella haemolytica. Cattle were infected with P. haemolytica via fiberoptic deposition of organisms into the posterior part of the right cranial lung lobe. Animals were euthanized at 2 or 4 h postinoculation (p.i.), and tissues were collected to assess PIC gene expression using antisense RNA probes specific for bovine interleukin-1α (IL-1α), IL-1β, IL-6, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) along with the β-actin (β-Act) housekeeping gene. Expression of PIC was induced at 2 h p.i. in P. haemolytica-infected cattle and continued to 4 h p.i. At 2 h p.i., induction of gene expression and increase of cells that expressed PIC were observed both in CD18+ and CD18− cattle after inoculation of P. haemolytica. The induction of gene expression with P. haemolytica inoculation was more prominent in CD18− cattle than in CD18+ cattle by comparison to pyrogen-free saline (PFS)-inoculated control animals. At 4 h p.i., however, the induction of PIC, especially IL-1α, IL-6, and IFN-γ, in the lungs of CD18+ cattle inoculated with P. haemolytica was greater than that in lungs of the CD18− cattle. IFN-γ and TNF-α genes were not increased in P. haemolytica-inoculated CD18− cattle lungs compared to the PFS-inoculated control lungs at 4 h p.i. In PFS-inoculated lungs, we generally observed a higher percentage of cells and higher level of gene expression in the lungs of CD18− cattle than in the lungs of CD18+ cattle, especially at 4 h p.i. The rate of neutrophil infiltration into the lungs of CD18− cattle at 2 h p.i. was significantly higher than that of CD18+ cattle; at 4 h p.i., there was no difference between the two groups. These data suggest that β2-integrins may contribute to the induction of expression of some PIC genes, as a consequence of P. haemolytica infection.
We identified two novel β-defensin precursors, preproGBD-1 and preproGBD-2, in the tissues of a goat. Although the precursors were identical in 96.8% of their bases and 88.2% (60 of 68) of their amino acids, preproGBD-1 was expressed principally in the tongue and respiratory tract, whereas preproGBD-2 expression predominated throughout the intestine. These findings exemplify the phenomenon of tissue-specific expression in a family of host defense peptides that arose before the avian and mammalian lineages diverged.
Affinity-purified rabbit polyclonal (PAB96-1) and mouse monoclonal (1G9-1C2) antibodies to synthetic H-DDDDDDD-OH, an antimicrobial anionic peptide (AP) originally isolated from ovine pulmonary surfactant, were prepared and used to assess the concentrations of AP-like molecules in human respiratory tract samples. In bronchoalveolar lavage fluids, concentrations of AP-like molecules measured by enzyme-linked immunosorbent assay were significantly lower in 13 patients with cystic fibrosis (CF) (mean ± standard deviation [SD], 0.78 ± 0.46 mM) than in 34 patients without CF (1.30 ± 0.66 mM) (P = 0.01). In pulmonary tissues of three patients without CF, very little antigen was stained in the apical cytoplasm of the bronchial and bronchiolar epithelium yet robust staining was seen in the alveolar epithelium. In pulmonary tissues of three patients with CF, robust staining of antigen was seen in the apical cytoplasm of the bronchial and bronchiolar epithelium yet no staining was seen in the alveolar epithelium. These results show that AP-like molecules are present in healthy human respiratory tract samples and differ in concentration and location of expression in patients with and without CF.
We purified three proline-rich antimicrobial peptides from elastase-treated extracts of sheep and goat leukocytes and subjected two of them, OaBac5α and ChBac5, to detailed analysis. OaBac5α and ChBac5 were homologous to each other and to bovine Bac5. Both exhibited potent, broad-spectrum antimicrobial activity under low-concentration salt conditions. While the peptides remained active against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Listeria monocytogenes in 100 mM NaCl, they lost activity against Staphylococcus aureus and Candida albicans under these conditions. ChBac5 was shown to bind lipopolysaccharide, a property that could enhance its ability to kill gram-negative bacteria. Proline-rich Bac5 peptides are highly conserved in ruminants and may contribute significantly to their innate host defense mechanisms.
Pasteurella haemolytica is an important respiratory pathogen of cattle that incites extensive infiltrates of neutrophils into the lung. In addition to the parenchymal damage caused by factors released by P. haemolytica, neutrophils contribute to the pathologic changes in the lungs. Molecules which mediate neutrophil infiltration into the lungs during P. haemolytica pneumonia are poorly characterized. To determine whether the CD18 family (β2-integrin) of leukocyte adhesion molecules mediates initial passage of neutrophils into the pulmonary bronchi and bronchioles of lungs infected with P. haemolytica, three Holstein calves homozygous for bovine leukocyte adhesion deficiency (BLAD) (CD18-deficient neutrophils), and three age- and breed-matched control calves (normal CD18 expression) were inoculated with P. haemolytica A1 via a fiberoptic bronchoscope and euthanized at 2 h postinoculation. Sections of lung were stained for neutrophils, and the intensity of neutrophilic infiltration was determined by computerized image analysis. Significantly fewer (P < 0.05) neutrophils infiltrated the lumen, epithelium, and adventitia of bronchioles and bronchi in lungs of calves with BLAD compared to normal calves, which had dense infiltrates within these sites at 2 h postinoculation. The reduced infiltration in calves with BLAD occurred despite the presence of an extremely large number of neutrophils in peripheral blood that is typical for these calves. The large number of neutrophils in the blood of calves with BLAD is probably a physiologic response that can occur without microbial colonization, since one calf with BLAD that was raised under germ-free conditions had large numbers of neutrophils in the blood that were similar to those in a calf with BLAD that was raised conventionally. Neutrophil counts in the germ-free and conventionally reared calves with BLAD were much higher than those in the three normal calves raised under germ-free conditions. The work in this study demonstrates that during the initial inflammatory response, neutrophils with normal CD18 expression pass more readily than CD18-deficient neutrophils into the walls and lumen of bronchi and bronchioles. It suggests that CD18 is needed for initial passage through the extensive extracellular matrix of the bronchi and bronchioles. This has potential importance for the development of therapies to direct or inhibit neutrophil infiltration into conducting airways rather than alveolar spaces.