Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Beta-defensin expression in human mammary gland epithelia.
Pediatr Res. 2000 Jul;48(1):30-5.
Milk of mammalian species contains a wide spectrum of anti-infectious factors, some of which are heat stable. Focusing on recently discovered heat-stable antibacterial peptides called defensins, which are expressed in epithelial tissues such as airway, skin, and kidney, we hypothesized that mammary gland epithelia produce and secrete defensins onto the epithelial surface and into milk. Using a reverse-transcription PCR assay, we identified the human beta-defensin-1 (hBD-1) gene transcript in a human mammary gland epithelial cell line, MCF-12A, and in mammary glandular tissue of nine nonlactating women. Epithelial cells harvested from milk of lactating women also expressed hBD-1 mRNA. Presence of hBD-1 peptide in mammary epithelia was confirmed by immunostaining with an hBD-1 antibody. In contrast, expression of human beta-defensin-2 was not apparent both at mRNA and protein levels. Our findings suggest a biologic role of hBD-1 in the human mammary gland.

Immunohistochemical staining of human alpha-defensin-1 (HNP-1), in the submandibular glands of patients with oral carcinomas.
Anticancer Res. 2000 Mar-Apr;20(2B):1125-7.
The purpose of this study was the immunohistochemical localization and distribution of HNP-1 in the submandibular glands of patients with oral carcinomas. Tissue sections were embedded in paraffin, and HNP-1 was immunostained by the streptavidin-biotin coupled peroxidase method. Striated duct cells in the submandibular glands were stained with anti-defensin antibody. Neutrophils and capillary intimal cells were also stained. Defensins (HNPs) are peptides that occur in neutrophils and protect against bacteria and tumor cells. Human alpha-defensin-1 (HNP-1) is such a peptide, possessing both antimicrobial and cytotoxic activities. The presence of HNP-1 in striated duct cells in the submandibular glands of oral cancer patients, suggests a likely role in tumor immunity, for this peptide.

Investigation of beta defensin gene expression in the ocular anterior segment by semiquantitative RT-PCR.
Br J Ophthalmol. 2000 May;84(5):523-6.
AIM: To determine if beta defensins are expressed in the anterior segment of the eye and to determine the temporal pattern of expression using a real time semiquantitative reverse transcription polymerase chain reaction (RT-PCR). METHODS: Ocular tissue (corneal epithelium, conjunctiva, iris, and lens capsule) was collected from 23 patients undergoing surgery. Serial corneal or conjunctival impression cytology was performed on a separate group of 10 patients undergoing corneal tunnel phacoemulsification or trabeculectomy. The samples were analysed for beta defensin mRNA by semiquantitative RT-PCR and the mRNA standardised for cell numbers. RESULTS: RT-PCR amplified beta defensin 1 mRNA from all lens capsule (six) and corneal (five) samples and all but one of the conjunctival (six) and iris samples (six). beta Defensin 2 mRNA was amplified from three of five corneal, two of six conjunctival, and none of the iris or capsule samples. The impression cytology samples demonstrated a decline in defensin expression over the three time points studied. There were no false positive results from either the no-RT or negative control samples. CONCLUSIONS: This preliminary study confirms that natural antibacterial peptides are expressed in the anterior segment of the eye. There appears to be a pattern to the expression with inducible beta defensin 2 not expressed intraocularly and higher levels of beta defensin 1 than beta defensin 2 expressed in extraocular tissue. The implication is that beta defensin 1 is constitutively produced in ocular tissues and represents a key component of the innate immune system.

Expression of human beta-defensin 1 mRNA in human nasal mucosa.
Acta Otolaryngol. 2000 Jan;120(1):58-61.
Antimicrobial peptides are cationic proteins that are found in a wide range of organisms. Recent reports suggested that human beta-defensin 1 (hBD-1), a prominent group of antimicrobial peptides, is an important component of the innate immune response, particularly at mucosal surfaces that are vulnerable to colonization by potential pathogens. Therefore, hBD-1 may participate in providing intrinsic nasal mucosal defence against microbial infections. The present study aimed to look for hBD-1 mRNA in human nasal mucosa without obvious signs of inflammation. Total RNA was isolated from human inferior turbinate mucosa and hBD-1 mRNA was detected in these tissues by using reverse transcription and polymerase chain reaction (RT-PCR). By in situ hybridization, hBD-1 mRNA was predominantly localized in superficial epithelial cells and submucosal glandular epithelium of human inferior turbinate mucosa. These data suggest that nasal epithelia and submucosal glands may secrete hBD-1, contributing to the mucosal defences of the nose.

Structural analysis of human beta-defensin-1 and its significance in urinary tract infection.
Nephron. 2000 May;85(1):34-40.
Human beta-defensin-1 (hBD-1) is a 36-amino-acid antimicrobial peptide that functions in the host innate defense. We developed a highly sensitive radioimmunoassay for hBD-1 and identified several hBD-1 peptides in human kidney, urine, and plasma by amino acid sequencing and mass spectrometry. Large quantities of hBD-1 peptides are produced in the kidney, are released into the tubular lumen as 47-amino-acid pro-hBD1, and then undergo proteolytic processing and generate multiple truncated forms. The respective urine and plasma concentrations of hBD-1 in patients with pyelonephritis are 48.1 +/- (SEM) 15.7 pmol/mg creatinine and 2.66 +/- 0.41 pmol/ml, 3.1-fold and 1.8-fold those of normal individuals. hBD-1 is thought to contribute to mucosal defense in the urinary tract. Our findings provide a better understanding of the biosynthesis of this peptide and its pathophysiological significance in infectious diseases.

Presence of defensin in epithelial Langerhans cells adjacent to oral carcinomas and precancerous lesions.
Anticancer Res. 1999 Jul-Aug;19(4B):2969-71.
We aimed to immunohistochemically study the localization of defensin (HNPs), a family of peptides with antimicrobial and cytotoxic activity, in oral tumor tissue. Therefore, tissue sections were embedded in paraffin, and defensin was immunostained by the streptavidin-biotin coupled peroxidase method. Langerhans cells were confirmed by indirect immunostaining with anti-S-100 protein polyclonal antibody. Melanocytes were stained with Fontana-Masson's stain. Neutrophils and intimal cells were stained by anti-defensin antibody. Langerhans cells in normal epithelium or dysplasic epithelium adjacent to squamous cell carcinoma and precancerous lesion were also stained. Defensins (HNPs) are nonspecific peptides that occur in neutrophils and protect against bacteria, fungi, and tumor cells. Since defensins are also found in epithelial Langerhans cells adjacent to tumor tissue, these peptides most likely have a role in tumor immunity.

Defensin-1, an antimicrobial peptide present in the saliva of patients with oral diseases.
Oral Dis. 1999 Apr;5(2):139-42.
OBJECTIVES AND DESIGN: A preceding paper has noted a detection of defensin-1 (HNP-1), a peptide with antimicrobial and cytotoxic properties, in the saliva of patients with oral squamous cell carcinoma. The present study deals with the presence of HNP-1 in the saliva of patients with various oral diseases. METHODS: Whole saliva samples were obtained from the patients. HNP-1 in the saliva was isolated and purified by HPLC and the amino acid sequence of the peptide was determined. The molecular weight of HNP-1 was measured by mass spectrometry. The concentration of HNP-1 in saliva was determined by comparing the height of eluted HNP-1 with that of a synthetic HNP-1 standard. RESULTS: The concentrations of HNP-1 in the saliva of patients with oral lichen planus (n = 5), leukoplakia (n = 4), and glossitis associated with iron deficiency (n = 4) were 8.3 +/- 4.3 micrograms ml-1, 13.2 +/- 7.9 micrograms ml-1, and 11.4 +/- 4.9 micrograms ml-1, (mean +/- s.d.), respectively. These concentrations were significantly higher than those in healthy subjects (0.8 microgram ml-1) (P < 0.01). In contrast, salivary HNP-1 concentrations in patients with glossodynia (n = 4) and oral discomfort (n = 4) were similar to those in healthy subjects. CONCLUSIONS: Since HNP-1 is a non-specific defensive peptide present in neutrophils, it may play an important role in the protection against diseases such as oral lichen planus, leukoplakia, and glossitis associated with iron deficiency.

Immunomagnetic recovery of human neutrophil defensins from the human gingival crevice.
Oral Microbiol Immunol. 1999 Jun;14(3):190-3.
The human neutrophilic protein defensins are cationic, antimicrobial, cytotoxic, corticostatic chemotactic, opsonic peptides found in azurophilic granules and constitute about 5% of the total protein in human neutrophils. In the present study, defensins were recovered from the human gingival crevice using paramagnetic microspheres (beads), coated with anti-defensin antibodies. The bead-bound defensins were assayed using an enzyme-linked immunosorbent assay developed in this laboratory. Twenty sites were sampled; defensin was found in 100% of the sites and ranged in amount from 270-2000 ng/site. The large local concentrations of defensins, estimated in the mg/ml range, may have major effects on the microbiology of the gingival crevice.

Human beta-defensin-1 mRNA is transcribed in tympanic membrane and adjacent auditory canal epithelium.
Infect Immun. 1999 Sep;67(9):4843-6.
The external auditory canal is less susceptible to infections than the sensitive middle-ear cavity. Since recent research has provided insight to the production of potent antimicrobial peptides from various surface epithelia, we wanted to investigate whether protection of the external auditory canal in part could be explained by the production of human beta-defensin-1 (HBD-1). This particular peptide is known to be constitutively expressed in various surface epithelia, such as airway, skin, and urogenital tissues. By reverse transcriptase PCR we demonstrate HBD-1 mRNA in the pars tensa and pars flaccida of the tympanic membrane and in the meatal skin. In situ hybridization studies localized the HBD-1 mRNA to the epidermal layer of these tissues. The HBD-1 transcripts were also evident in the sebaceous glands and in hair follicles of the meatal skin. In contrast, HBD-1 mRNA was not detected in the tympanal epithelium of the eardrum. The widespread presence of mRNA encoding for this broad-spectrum antimicrobial peptide in the meatal skin and tympanic membrane suggests that HBD-1 participates in the innate antimicrobial defense of the external auditory canal and middle-ear cavity.

Reduced antimicrobial peptide expression in human burn wounds.
Burns. 1999 Aug;25(5):411-3.
Severely burned skin ceases to perform its natural protective role and surrenders itself as a nidus and portal for bacterial invasion. Antimicrobial peptides are part of a non-specific chemical defence system, separate from cellular and humoral immunity. Two of these peptides, human beta-defensins 1 and 2 have been recently found in skin and are produced by keratinocytes. Beta defensins have potent bactericidal activity against a wide spectrum of bacterial and fungal organisms commonly responsible for burn wound infections. To date, expression of beta defensins has not been examined in the human burn wound. Our findings demonstrate that expression of hBD-2 is greatly decreased in the burn wound whereas hBD-1 appears to be preserved. These results may have important implications in the pathogenesis and treatment of invasive burn sepsis.