Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Inhibition of recombinant adeno-associated virus (rAAV) transduction by bronchial secretions from cystic fibrosis patients.
Gene Ther. 2000 Oct;7(20):1783-9.
The conducting airways are the primary target for gene transfer in cystic fibrosis (CF), yet the inflammation associated with CF lung disease could potentially pose a significant barrier to gene transfer vectors, such as recombinant adeno-associated virus (rAAV). In order to investigate this possibility, aliquots of bronchoalveolar lavage (BAL) fluid from eight individuals with CF were tested for their in vitro inhibitory effects on rAAV transduction, along with BAL from non-CF individuals. While the non-CF BAL fluid was not inhibitory, seven of eight CF BAL samples had significant inhibitory activity, resulting in a five- to 20-fold reduction in transduction events. Inhibition of rAAV transduction by CF BAL could be reversed by alpha-1-antitrypsin (AAT), but not by DNase. When neutrophil elastase and neutrophil alpha defensins (human neutrophil peptides, HNP) were measured in these samples, they were elevated by 500- and 10,000-fold, respectively. The levels of HNP correlated inversely with the amount of rAAV transduction. Furthermore, rAAV transduction could be blocked by purified HNP in an AAT-reversible manner at HNP concentrations within the range measured in these fluids. We conclude that products of inflammation in CF BAL fluid are inhibitory to rAAV transduction, and that these effects may be reversible by AAT.

Synergistic and additive killing by antimicrobial factors found in human airway surface liquid.
Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L799-805.
Airway surface liquid contains multiple factors thought to provide a first line of defense against bacteria deposited in the airways. Although the antimicrobial action of individual factors has been studied, less is known about how they work in combination. We examined the combined action of six antimicrobial peptides found in airway surface liquid. The paired combinations of lysozyme-lactoferrin, lysozyme-secretory leukocyte protease inhibitor (SLPI), and lactoferrin-SLPI were synergistic. The triple combination of lysozyme, lactoferrin, and SLPI showed even greater synergy. Other combinations involving the human beta-defensins, LL-37, and tobramycin (often administered to cystic fibrosis patients by inhalation) were additive. Because the airway surface liquid salt concentration may be elevated in cystic fibrosis patients, we examined the effect of salt on the synergistic combinations. As the ionic strength increased, synergistic interactions were lost. Our data suggest that the antibacterial potency of airway surface liquid may be significantly increased by synergistic and additive interactions between antimicrobial factors. These results also suggest that increased salt concentrations that may exist in cystic fibrosis could inhibit airway defenses by diminishing these synergistic interactions.

Human beta defensin is absent in burn blister fluid.
Burns. 2000 Dec;26(8):724-6
Defensins are a family of cationic antimicrobial peptides that participate in innate host defence. Human beta defensin-2 (HBD-2) is produced by human keratinocytes, and has a potent bactericidal activity against a wide spectrum of microorganisms. We have recently shown that expression of HBD-2 is present in normal skin and lost in the full-thickness burn wound. Defensins have been found in the blister fluid of chronic wounds. Our study was designed to examine blister fluid from partial-thickness burns for defensin content. Fluid from five patients was collected from partial-thickness burn blisters, and then analysed by sandwich Enzyme-Linked Immunosorbent Assay (ELISA) with a monoclonal antibody and rabbit polyclonal antibody to HBD-2. The assay was validated against a Western blot assay for HBD-2 in samples of bronchoalveolar lavage fluid from patients with inflammatory lung disease. No HBD-2 was detectable in any of the burn blister fluids analysed. HBD-2 is lost in the full-thickness burn wound, and we have now demonstrated its absence in burn blister fluid. This finding represents evidence of a host defence defect within the burn wound and suggests a possible therapeutic role for antimicrobial peptides in the management of burn wounds.

Expression of the antimicrobial peptide, human beta-defensin 1, in duct cells of minor salivary glands and detection in saliva.
J Dent Res. 2000 Sep;79(9):1669-74.
The oral cavity is exposed to a variety of environmental insults. Salivary secretions play a critical role in maintaining oral health via innate host defense mechanisms and secretion of secretory IgA. Human beta-defensins (hBD) are antimicrobial peptides that are a component of the innate immune response; they are expressed in epithelia and are proposed to have a role in mucosal defense. hBD-1 mRNA is constitutively expressed in numerous mucosal tissues, including human gingiva and submandibular and parotid glands. Our objective was to detect the expression and localization of hBD-1 peptide in human salivary glands and in saliva. Minor salivary gland tissue was obtained from biopsies of patients with mucoceles (n = 20). hBD-1 peptide was detected by immunohistochemistry; expression was localized to the ductal cells and not the acinar cells of these glands. The peptide was located apically, toward the lumen in the duct cells. Further evaluation showed stronger hBD-1 expression in ducts with periductal inflammation, as indicated by the immunostaining of serial sections with anti-CD45 specific for B- and T-lymphocytes. Statistical analysis showed a strong correlation of hBD-1 staining and inflammation. Results of immunolocalization suggest that hBD-1 functions to protect salivary glands from retrograde infection, that expression of the peptide is enhanced in inflamed sites, and that post-transcriptional regulatory mechanisms may be involved in hBD-1 peptide expression. Western immunoblot analysis also detected hBD-1 peptide in unstimulated, whole, acidified saliva from normal volunteers. However, hBD-1 peptide associated with salivary mucin resulted in loss of the detection in a dot-immunoblot assay. Association of hBD-1 with salivary mucin may facilitate peptide distribution and adherence to oral surfaces and aid its function within the oral cavity.

Expression of human beta-defensins in intraocular tissues.
Invest Ophthalmol Vis Sci. 2000 Sep;41(10):3026-31.
PURPOSE: Defensins are naturally occurring antimicrobial peptides. Recently the authors published evidence of defensin production by the human ocular surface. A study was undertaken to look for intraocular defensins that may account for unexplained antimicrobial activity of intraocular fluids. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was performed on human postmortem ciliary body samples for beta defensins-1 (HBD-1) and beta defensin-2 (HBD-2), and alpha defensins 5 and 6. Induction of defensins by cytokines was analyzed in cultured human ciliary body epithelial (CBE) and retinal pigment epithelial (RPE) cells. Polyclonal antibodies were used to immunoblot aqueous and vitreous to detect HBD-1 and HBD-2 and to estimate their concentration. RESULTS: RT-PCR revealed constitutive HBD-1 message in ciliary body. HBD-2 and alpha defensin 5 and 6 messages were absent. HBD-2 message was induced by cytokine stimulation of both CBE and RPE cells. Immunoblots of vitreous and aqueous stained positively for HBD-1 but not HBD-2. The estimated aqueous concentration of HBD-1 was less than 16 ng/ml. CONCLUSIONS: This study demonstrates that HBD-1 is constitutively present in the aqueous and vitreous, probably at sub-bacteriocidal concentrations. HBD-2 was absent from aqueous, but cytokine stimulation studies suggest that it may be generated in response to inflammatory cytokines during infections. HBD-2 has a wider antibacterial spectrum, is 10-fold more potent, and may play a more significant role in antimicrobial defense than HBD-1. The use of defensins therapeutically may be indicated; however, caution is required because defensins also promote cell proliferation and fibrin formation, which are 2 key elements in ocular scarring processes such as proliferative vitreoretinopathy.

Differential expression of human alpha- and beta-defensins mRNA in gastrointestinal epithelia.
Eur J Clin Invest. 2000 Aug;30(8):695-701.
BACKGROUND: While defensins have received great attention for their role in bronchial innate immune defence, little is known about the expression levels of the four human epithelial defensins (HD5, HD6, hBD1 and hBD2) in the digestive tract. In this study we quantified the alpha- and beta-defensins mRNA in biopsies obtained from the gastrointestinal mucosa and identified the cells expressing the beta-defensin hBD1 mRNA in ileal mucosa. MATERIAL AND METHODS: Biopsies from human stomach (corpus and antrum), duodenum, jejunum, ileum and colon were analysed for their expression of alpha- and beta-defensins. The mRNA of defensins was quantified by semiquantitative reverse transcription-polymerase chain reaction. Cells expressing beta-defensin hBD1 mRNA were identified by in situ hybridization with 35S-labelled RNA probes in tissue sections of human ileum. RESULTS: The hBD1 mRNA was expressed at low levels with little variability throughout the gastrointestinal tract and was detected in all epithelial cells of ileal mucosa. HD5 and HD6 mRNA expression was restricted to the intestine and displayed high interindividual variability. The highest expression levels were observed in jejunum and ileum. Biopsies obtained from duodenum displayed low levels or no expression of HD5 and HD6. The expression level increased considerably in a biopsy obtained from a patient with acute coeliac sprue. In contrast, low levels were observed in a biopsy from a patient with coeliac sprue in remission. CONCLUSIONS: The expression levels of hBD1, HD5 and HD6 throughout the gastrointestinal tract are tissue and peptide specific and these defensins are expressed with high interindividual variability.

Determination of defensin HNP-1, HNP-2, and HNP-3 in human saliva by using LC/MS.
Peptides. 2000 Jun;21(6):757-65.
The mass spectral profiling of saliva by liquid chromatography mass spectrometry in relation to particular types of pain is being examined. The aim is to develop a profile that could be useful for the assessment of patients and their treatment programs, as well as identifying unknown compounds observed in saliva. Defensin human neutrophil peptide-1 (HNP-1) and defensin HNP-2 were identified and confirmed, whereas defensin HNP-3 was tentatively identified. Linear calibration range of defensin HNP-1 and HNP-2 was 0.25 to 3 microg/ml with R(2) values of > 0.99 for both. The detection limit for defensin HNP-1 and HNP-2 was estimated at 0.1 microg/ml. The healthy subjects surveyed in this study had readily measurable salivary concentrations of defensin HNP-1 (8.6 +/- SD 8.0 microg/ml) and defensin HNP-2 (5.6 +/- SD 5.2 microg/ml).

Presence of human beta-defensin-2 in oral squamous cell carcinoma.
Anticancer Res. 2000 May-Jun;20(3B):2005-7.
The purpose of this study was to demonstrate immunohistochemically the localization and distribution of human beta-defensin-2 (HBD-2), a peptide with antimicrobial activity, in oral carcinoma tissues. Tissue samples were embedded in paraffin, and HBD-2 was immunostained by the streptavidin-biotin coupled peroxidase method. Cancer cells in the cornified region of well differentiated squamous cell carcinomas were stained intensely. Stained cancer cells detected by anti-HBD-2 antibody were scattered among the cells of the non-cornified region.

Expression of beta-defensin genes by human salivary glands.
Oral Microbiol Immunol. 1999 Dec;14(6):371-4.
This study investigated expression of genes encoding human beta-defensins 1 and 2 by human salivary glands. Tissues from surgical biopsies were collected fresh onto ice and stored in liquid nitrogen. Total RNA was extracted using Trizol reagent and human beta-defensin messenger RNA detected by reverse transcriptase polymerase chain reaction amplification. DNA sequencing of amplified fragments, after ligation into pGEM-T Easy vector and transformation of competent Escherichia coli, confirmed identities of cloned fragments. Human beta-defensin 1 messenger RNA was detected in all 25 samples that generated amplifiable cDNA, as assessed using abl-specific primers. Three of 13 submandibular gland samples (two normal, one chronically inflamed), and 2 of 2 minor salivary gland samples (one normal, one chronically inflamed) expressed human beta-defensin 2 messenger RNA. All six parotid gland samples studied were negative for human beta-defensin 2 messenger RNA. Thus, human beta-defensin 1 gene expression occurred in all human major and minor salivary glands studied, whereas human beta-defensin 2 expression occurred only in a small number of gland samples.

Detection of human alpha-defensin-1, an antimicrobial peptide, in the fluid of jaw cysts.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000 Jul;90(1):78-81.
OBJECTIVE: The purpose of this study was to isolate and to identify an antimicrobial peptide, human alpha-defensin-1 (HNP-1) in jaw cyst fluid. STUDY DESIGN: We collected jaw cyst fluid from 22 patients with various jaw cysts and analyzed the peptide components of them by reversed-phase high-performance liquid chromatography (HPLC). HNP-1 in the jaw cyst fluid was identified by the amino acid sequence and the molecular weight. The concentration of HNP-1 in the fluid of various jaw cysts was quantified by HPLC. RESULTS: HNP-1 in the jaw cyst fluid was isolated, purified, and identified. The concentrations of HNP-1 in the jaw cyst fluid ranged from 27.0 to 3725.4 microg/mL. CONCLUSION: HNP-1 was detected in the fluid of various types of jaw cysts.