Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
A single-nucleotide polymorphism in the human beta-defensin 1 gene is associated with HIV-1 infection in Italian children.
AIDS. 2004 Jul 23;18(11):1598-600.
In this study we show a significant correlation between a single-nucleotide polymorphism in the 5'-untranslated region of the DEFB1 gene, which probably regulates the gene expression of human beta defensin 1 (hBD-1) and the risk of HIV-1 infection in an Italian paediatric population (97 HIV-1 perinatally infected children), pointing to the importance of innate immunity in HIV-1 infection.

High concentrations of alpha-defensins in plasma and bronchoalveolar lavage fluid of patients with acute respiratory distress syndrome
Life Sci. 2004 Jul 16;75(9):1123-34
alpha-Defensins, antimicrobial peptides localized in neutrophils, participate in tissue damage through their cytotoxic effects in neutrophil-mediated pulmonary diseases. Neutrophils play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS). We measured alpha-defensins levels in plasma and bronchoalveolar lavage fluid (BALF) of ARDS patients to assess the kinetics of alpha-defensins in ARDS. Plasma alpha-defensins levels were higher in ARDS patients than in control subjects, and BALF levels were also higher in ARDS patients than in control subjects. In ARDS, BALF alpha-defensins levels correlated with those of interleukin (IL)-8, and plasma alpha-defensins levels also correlated with Lung Injury Score. Peripheral neutrophil alpha-defensins contents were higher in ARDS than the control. IL-8 dose-dependently stimulated alpha-defensins release from cultured neutrophils and these levels were higher in ARDS than the control. Reverse-phase high performance liquid chromatography showed high plasma levels of pro-defensins, precursors of alpha-defensins from the bone marrow in ARDS, although alpha-defensins in peripheral and BALF neutrophils were mature type. In conclusion, high plasma alpha-defensins in ARDS patients result from the release of pro-defensins from bone marrow, rather than mature alpha-defensins from neutrophils that accumulate in the alveolar space. The alpha-defensins contents of peripheral neutrophils in ARDS are higher and easier to release than control.

Expression of human beta-defensins-1 and -2 peptides in unresolved chronic periodontitis.
J Periodontal Res. 2004 Aug;39(4):221-7.
BACKGROUND: Human beta-defensins (hBDs) are antimicrobial peptides which contribute to host innate immunity by disrupting the membrane integrity of a broad spectrum of microorganisms. OBJECTIVES: This study aimed to determine the expression profiles of hBD-1 and -2 peptides in gingiva and to assess the possible relations of these antimicrobial peptides with periodontal health and disease. METHODS: Seven periodontally healthy subjects and 22 patients with unresolved chronic periodontitis were recruited and the gingival biopsies collected consisted of healthy tissues from the healthy subjects (HT-C); periodontal pocket tissues (PoT) and inflamed connective tissues (ICT) from the base of pocket, i.e. granulation tissues, as well as clinically healthy tissues (HT-P) from the adjacent clinically healthy sites from the patients. The expression of hBD-1 and -2 peptides was detected by immunohistochemistry and quantitatively analyzed with a computerized image processing system. RESULTS: Both hBD-1 and -2 peptides were detected in all periodontally healthy subjects, while hBD-1 was detected in all patients and hBD-2 was found in most of the patients. Their expression was mainly confined to the granular and spinous layers of gingival epithelium, in which hBD-1 was detected in both intercellular spaces and cytoplasm, whereas hBD-2 was mainly observed in the cytoplasm. HT-C expressed significantly higher levels of hBD-2 than HT-P (p < 0.05). Within the patients, both defensins were up-regulated significantly in PoT as compared with the adjacent HT-P (p < 0.05). CONCLUSIONS: The present study showed that hBD-1 and -2 were frequently expressed in the granular and spinous layers of gingival epithelia and their expression may be associated with periodontal health and disease.

Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease
Am J Gastroenterol. 2004 May;99(5):894-904
OBJECTIVES: Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated. METHODS: Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry. RESULTS: Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), alpha-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-gamma mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. beta-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected. CONCLUSIONS: Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-gamma production by intraepithelial lymphocytes.

Human alpha defensin in HIV-exposed but uninfected individuals
J Acquir Immune Defic Syndr. 2004 Apr 15;35(5):455-63
Human alpha defensins 1, 2, and 3 are produced by CD8 T cells of HIV-infected long-term nonprogressors and have an antiviral activity. alpha Defensins were examined in peripheral blood mononuclear cells (PBMCs), cervical-vaginal mononuclear cells (CVMCs), and cervical biopsies of 9 HIV-1-exposed but uninfected women (ESNs), 10 HIV-infected patients (HIV), and 13 low-risk healthy controls (HCs). Results showed that, whereas alpha defensin production and alpha defensin-expressing CD8 lymphocytes were comparable in ESNs and HIV patients, constitutive alpha defensin production by peripheral CD8 and CVMCs was augmented in ESNs compared with HCs (P = 0.001 and P = 0.058, respectively); alpha defensin mRNA was increased in PBMCs of ESNs; unstimulated, alpha defensin-expressing peripheral and mucosal CD8 lymphocytes were 10-fold higher in ESNs compared with HCs (P = 0.003 and P = 0.01, respectively); and alpha defensin mRNA and alpha defensin-expressing cells were augmented in cervical biopsies of ESN compared with HCs (mRNA:P = 0.03). The differences were reduced upon in vitro mitogen stimulation. A robust constitutive production of alpha defensin is seen in HIV-exposed uninfected individuals; these peptides could have a role in the potentially protective immune response that characterizes ESNs.

Decreased excretion of antimicrobial proteins and peptides in saliva of patients with oral candidiasis.
J Oral Pathol Med. 2003 Nov;32(10):586-94.
BACKGROUND: Antimicrobial peptides in saliva appear to play a crucial role in the regulation of oral Candida growth, and study on antimicrobial excretion in saliva and oral candidiasis appears useful for the analysis of pathophysiology of oral candidiasis. METHODS: To clarify the role of saliva in the regulation of oral Candida growth, the levels of antimicrobial proteins and peptides and their excretion rates were examined in saliva obtained from 50 patients with oral candidiasis and 35 healthy individuals. RESULTS: The inhibitory activities of patients' saliva against Candida adhesion with HeLa cells and against Candida growth (radiolabeled glucose incorporation) were lower than those of saliva from the healthy controls. The salivary levels of lactoferrin (Lf; 11 +/- 9 microg/ml), secretory immunoglobulin A (sIgA; 160 +/- 37 microg/ml), beta-defensin 1 (375 +/- 37 ng/ml), and beta-defensin 2 (412 +/- 51 ng/ml) in the patients were largely lower than those in the control group (33 +/- 14 microg/ml, 204 +/- 51 microg/ml, 452 +/- 89 ng/ml, and 530 +/- 142 ng/ml, respectively), although the transferrin (Tf) and secretory component (SC) levels were almost same in both groups, and alpha-defensin 1 was slightly increased in the patient group (660 +/- 115 ng/ml vs. 467 +/- 168 ng/ml). In addition, the excretion rates of the proteins and peptides were largely decreased in the patients (Tf: 14 +/- 2 microg/10 min vs. 34 +/- 7 microg/10 min; Lf: 18 +/- 11 microg/10 min vs. 139 +/- 43 microg/10 min; sIgA: 300 +/- 132 microg/10 min vs. 900 +/- 207 microg/10 min; SC: 112 +/- 46 microg/10 min vs. 292 +/- 64 microg/10 min; alpha-defensin 1: 1223 +/- 431 ng/10 min vs. 2044 +/- 612 ng/10 min; beta-defensin 1: 687 +/- 243 ng/10 min vs. 1985 +/- 295 ng/10 min; and beta-defensin 2: 784 +/- 299 ng/10 min vs. 2288 +/- 278 ng/10 min). CONCLUSION: These results conclusively suggest that oral candidiasis is associated with salivary gland hypofunction and that decreases of salivary antibacterial proteins induce Candida overgrowth.

Expression of human beta-defensin 2 in human nasal mucosa
Eur Arch Otorhinolaryngol. 2004 May;261(5):238-41
Human beta-defensin (HBD)-2, an antimicrobial peptide, has been discovered to be produced by a number of epithelial cells. It is identified as a key element in the innate host defense mechanism. Because little is known about the expression of HBD-2 in the human sinonasal tract, we conducted this study to investigate the expression of the HBD-2 mRNA gene by the reverse transcription polymerase chain reaction (RT-PCR) and localization of HBD-2 peptide by immunohistochemistry in human nasal inferior turbinates and nasal polyps. RT-PCR showed significantly higher expression of HBD-2 mRNA in nasal polyps than in inferior turbinates. Using immunohistochemistry, HBD-2 peptide was predominantly localized in surface epithelial cells. Thus, it is feasible that HBD-2 is expressed in nasal mucosa and is upregulated in a condition of chronic inflammation.

Cytokine milieu of atopic dermatitis, as compared to psoriasis, skin prevents induction of innate immune response genes.
J Immunol. 2003 Sep 15;171(6):3262-9
Atopic dermatitis (AD) and psoriasis are the two most common chronic skin diseases. However patients with AD, but not psoriasis, suffer from frequent skin infections. To understand the molecular basis for this phenomenon, skin biopsies from AD and psoriasis patients were analyzed using GeneChip microarrays. The expression of innate immune response genes, human beta defensin (HBD)-2, IL-8, and inducible NO synthetase (iNOS) was found to be decreased in AD, as compared with psoriasis, skin (HBD-2, p = 0.00021; IL-8, p = 0.044; iNOS, p = 0.016). Decreased expression of the novel antimicrobial peptide, HBD-3, was demonstrated at the mRNA level by real-time PCR (p = 0.0002) and at the protein level by immunohistochemistry (p = 0.0005). By real-time PCR, our data confirmed that AD, as compared with psoriasis, is associated with elevated skin production of Th2 cytokines and low levels of proinflammatory cytokines such as TNF-alpha, IFN-gamma, and IL-1beta. Because HBD-2, IL-8, and iNOS are known to be inhibited by Th2 cytokines, we examined the effects of IL-4 and IL-13 on HBD-3 expression in keratinocyte culture in vitro. We found that IL-13 and IL-4 inhibited TNF-alpha- and IFN-gamma-induced HBD-3 production. These studies indicate that decreased expression of a constellation of antimicrobial genes occurs as the result of local up-regulation of Th2 cytokines and the lack of elevated amounts of TNF-alpha and IFN-gamma under inflammatory conditions in AD skin. These observations could explain the increased susceptibility of AD skin to microorganisms, and suggest a new fundamental rule that may explain the mechanism for frequent infection in other Th2 cytokine-mediated diseases.

Expression of human beta-defensins in conjunctival epithelium: relevance to dry eye disease
Invest Ophthalmol Vis Sci. 2003 Sep;44(9):3795-801
PURPOSE: The goals of this study were to investigate whether beta-defensins are differentially expressed in the conjunctival epithelium of patients with moderate dry eye when compared with normal subjects and whether proinflammatory cytokines or bacteria can modulate the expression of human beta-defensins (hBDs)-1, -2, and -3 by conjunctival epithelial cells. METHODS: RNA extracted from conjunctival impression cytology specimens of eight normal subjects and nine patients with moderate dry eye was used in RT-PCR to detect mRNA for hBDs-1, -2, and -3. Two conjunctival epithelial cell lines and primary cultured conjunctival epithelial cells were treated with proinflammatory cytokines or heat-killed Pseudomonas aeruginosa. RT-PCR and immunoblot analysis were used to detect mRNA for hBD-1, -2, and -3 and protein secretion of hBD-2, respectively. RESULTS: hBD-2 message was detected in RNA samples of eight of nine patients with dry eye, but not in any of the normal subjects' samples, whereas hBD-1 and -3 were detected in all subjects tested. RT-PCR revealed an upregulation of hBD-2 but no difference in expression of hBD-1 and -3 in cultured conjunctival cells after a 24-hour treatment with 10 ng/mL interleukin (IL)-1beta, IL-1beta and tumor necrosis factor-alpha (10 ng/mL) or heat-killed Pseudomonas aeruginosa (1 million colony-forming units; n = 3). hBD-2 expression was upregulated from 4 hours of treatment with IL-1beta (at 10 ng/mL; (n = 2-3) and at a concentration of 0.1 ng/mL IL-1beta (24-hour treatment; n = 2-3). Immunoblots demonstrated protein secretion results corresponding to the RT-PCR data. CONCLUSIONS: hBD-2 was expressed only in the conjunctival epithelium of patients with moderate dry eye. Because cytokines such as IL-1beta and TNF-alpha induced the expression of hBD-2 by conjunctival epithelial cells and because increased proinflammatory cytokine activity is a feature of dry eye disease, it can be speculated that the hBD-2 upregulation observed in subjects with moderate dry eye is mediated by proinflammatory cytokine activity.

Intraarticular release and accumulation of defensins and bactericidal/permeability-increasing protein in patients with rheumatoid arthritis.
J Rheumatol. 2003 Aug;30(8):1719-24.
OBJECTIVE: Defensins and bactericidal/permeability-increasing protein (BPI) are the components of the azurophilic granules of polymorphonuclear cells (PMNC) maintaining antimicrobial protection. Both these substances have been suggested to interact with the host immune system rather than merely kill invading pathogens. We assessed concentrations of BPI and a-defensins in synovial fluid (SF) and matching blood samples of patients with rheumatoid arthritis (RA). METHODS: Matching samples of SF and blood were collected from 67 patients with RA (aged 21-73 yrs) with acute joint effusion. Blood samples from 22 healthy individuals made up a control group. Concentrations of BPI and human neutrophil peptides (HNP 1-3) were measured by ELISA. The results were related to radiological signs of destructive arthritis, duration of the disease, and laboratory markers of inflammation. RESULTS: BPI and HNP concentrations in SF were 10-60 times higher than in matching blood samples (p < 0.0001). Strong correlations between BPI and HNP concentrations were found in both blood and SF. In SF, BPI and HNP concentrations correlated to white blood cell (WBC) count (p < 0.001), and were associated with erosive joint disease (p < 0.05). In contrast, WBC count, serum C-reactive protein, or rheumatoid factor were not significantly correlated to the BPI or HNP concentrations. Serum BPI concentrations were moderately but significantly increased in RA patients compared in blood to controls (p < 0.05). CONCLUSION: BPI and HNP are accumulated in the synovial cavity of patients with RA. Significant correlation between joint erosion and local occurrence of BPI and HNP suggests participation of these molecules in regulation of the destructive course of RA.