Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Antimicrobial Peptide immunity protects human nasal and auricular cartilage against infection.
J Craniofac Surg. 2010 Jan;21(1):198-201.
BACKGROUND:: Despite being impervious to surveillance by the adaptive immune system because of its lack of vascularity, infection of the nasal and auricular cartilage after surgery such as rhinoplasty or otoplasty is rare. Why is this so? Our goal was to determine whether the expression of antimicrobial peptides provides a previously unrecognized nonepithelial layer of innate immune defense within the nasal and auricular cartilage. MATERIALS AND METHODS:: Seven samples of nasal septum cartilage and 2 biopsies from auricular cartilage grafts were harvested during rhinoplasty and otoplasty procedures. Ten cadaveric samples of auricular and 9 samples of nasal cartilage were also obtained. Immunohistochemical staining was directed against the human beta-defensin antimicrobial peptides (hBD) 1, 2, and 3. A semiquantitative analysis was performed to measure immunoreactivity. RESULTS:: All 3 human beta-defensins were detected along the perichondral line and within the cartilage matrix in the nasal and auricular samples. Areas with positive immunohistochemical staining were also detected within chondrocyte cytoplasm. CONCLUSIONS:: We provide the first evidence of antimicrobial peptide expression (hBD-1, -2 and -3) within the perichondrium and cartilage matrix layers of the nasal and auricular cartilage. This previously unrecognized innate immune function of perichondrocytes and chondrocytes may explain the resistance of the nasal and auricular cartilage to infection after surgical procedures despite the absence of a vascular system.

Mucosal gene expression of antimicrobial peptides in inflammatory bowel disease before and after first infliximab treatment.
PLoS One. 2009 Nov 24;4(11):e7984.
BACKGROUND: Antimicrobial peptides (AMPs) protect the host intestinal mucosa against microorganisms. Abnormal expression of defensins was shown in inflammatory bowel disease (IBD), but it is not clear whether this is a primary defect. We investigated the impact of anti-inflammatory therapy with infliximab on the mucosal gene expression of AMPs in IBD. METHODOLOGY/PRINCIPAL FINDINGS: Mucosal gene expression of 81 AMPs was assessed in 61 IBD patients before and 4-6 weeks after their first infliximab infusion and in 12 control patients, using Affymetrix arrays. Quantitative real-time reverse-transcription PCR and immunohistochemistry were used to confirm microarray data. The dysregulation of many AMPs in colonic IBD in comparison with control colons was widely restored by infliximab therapy, and only DEFB1 expression remained significantly decreased after therapy in the colonic mucosa of IBD responders to infliximab. In ileal Crohn's disease (CD), expression of two neuropeptides with antimicrobial activity, PYY and CHGB, was significantly decreased before therapy compared to control ileums, and ileal PYY expression remained significantly decreased after therapy in CD responders. Expression of the downregulated AMPs before and after treatment (DEFB1 and PYY) correlated with villin 1 expression, a gut epithelial cell marker, indicating that the decrease is a consequence of epithelial damage. CONCLUSIONS/SIGNIFICANCE: Our study shows that the dysregulation of AMPs in IBD mucosa is the consequence of inflammation, but may be responsible for perpetuation of inflammation due to ineffective clearance of microorganisms.

In vivo release of alpha-defensins in plasma, neutrophils and CD8 T-lymphocytes of patients with HIV infection.
Curr HIV Res. 2009 Nov;7(6):650-5.
alpha-Defensins are reported to be a soluble component of innate immunity actively participating in host defense against HIV. In order to further investigate the role of alpha-defensins in innate immunity during HIV infection, we analyzed CD8+ T lymphocytes and neutrophils obtained from 34 HIV-infected and 14 uninfected subjects. CD8+ T cells and neutrophils were labelled for evaluating alpha-defensin expression by flow cytometric analysis using a dual laser FACScalibur. Culture supernatants and plasma were also collected for ELISA quantification of alpha-defensins. The results showed a significantly increased production of alpha-defensins in plasma, neutrophils and CD8 T-lymphocytes of patients with HIV infection in comparison with healthy controls. The expression of alpha-defensins, by CD8+ cells probably reflects both the intrinsic production and the uptake from cocultured cells that release defensins. The upregulation of alpha-defensin expression within neutrophils could account for the increased release of such peptides in the systemic circulation. Antiretroviral treatment did not have any effect on plasma levels and expression of alpha-defensins by neutrophils. Overall, our findings suggest that the increased production/expression of alpha-defensins could be correlated with the chronic process of immune activation seen in HIV infection.

Differential expression pattern of genes encoding for anti-microbial peptides in the fetal membranes of patients with spontaneous preterm labor and intact membranes and those with preterm prelabor rupture of the membranes.
J Matern Fetal Neonatal Med. 2009 Dec;22(12):1103-15.
OBJECTIVE: Increased amniotic fluid concentrations of anti-microbial peptides, components of the innate immune system, have been reported in patients with preterm labor (PTL) with intact membranes and intra-amniotic infection and/or inflammation (IAI), as well as in patients with preterm prelabor rupture of the membranes (PPROM). This study was designed to confirm these results using a targeted approach, detecting DEFA1, DEFB1, GNLY, and S100A9 gene expression in the choriamniotic membranes in pregnancies complicated with PTL and intact membranes or PPROM, with and without histologic chorioamnionitis. STUDY DESIGN: Human fetal membranes were obtained from patients in the following groups: (1) PTL with intact membranes (n = 15); (2) PTL with intact membranes with histologic chorioamnionitis (n = 12); (3) PPROM (n = 17); and (4) PPROM with histologic chorioamnionitis (n = 21). The mRNA expression of alpha-defensin-1, beta-defensin-1, calgranulin B and granulysin in the fetal membranes was determined by qRT-PCR. RESULTS: (1) The expression of alpha-defensin-1 mRNA in the fetal membranes was higher in patients with PTL and intact membranes with histologic chorioamnionitis, than those without chorioamnionitis (19.4-fold, p < 0.001); (2) Among patients with histologic chorioamnionitis, patients with PTL and intact membranes had a higher alpha-defensin-1 mRNA expression than those with PPROM (5.5-fold, p = 0.003); (3) Histologic chorioamnionitis was associated with a higher calgranulin B mRNA expression in the chorioamniotic membranes of patients with both PTL and intact membranes (7.9-fold, p = 0.03) and PPROM (7.6-fold, p < 0.0001); (4) The expression of calgranulin B mRNA in the fetal membranes was higher in patients with PTL and intact membranes without histologic chorioamnionitis than in those with PPROM without histologic chorioamnionitis (2.7-fold, p = 0.03); (5) There were no differences in the expression of beta-defensin-1 and granulysin in the chorioamniotic membranes between the study groups even in the presence of histologic chorioamnioniotis. CONCLUSIONS: (1) Among patients with histologic chorioamnionitis, the mRNA expression of alpha-defensin-1 and calgranulin B in the fetal membranes of patients with PTL and intact membranes as well as that of calgranulin B in the fetal membranes of patients with PPROM is higher than in the membranes of those without histologic chorioamnionitis; (2) histologic chorioamnionitis is associated with differences in the pattern of alpha-defensin-1 mRNA expression in the fetal membranes in patients with PTL and intact membranes and those with PPROM.

Defective Paneth cell-mediated host defense in pediatric ileal Crohn's disease.
Am J Gastroenterol. 2010 Feb;105(2):452-9.
OBJECTIVES:Adult ileal Crohn's disease (CD) is characterized by a specific decrease in ileal Paneth cell alpha-defensins. In addition to NOD2, we previously identified a disturbance of the Wnt-signaling transcription factor TCF-4 as a major mechanism for this deficiency. The aim of this study was to evaluate human alpha-defensin-5 (HD-5) and TCF-4 in an independent cohort of pediatric CD patients.METHODS:Expression levels of HD-5 and TCF-4 mRNA were quantified by real-time PCR in biopsies from newly diagnosed untreated pediatric CD patients (<18 years, n=36) and age-matched symptomatic non-inflammatory bowel disease controls with a histologically normal gut (n=29). To assess the influence of current inflammation, mucosal interleukin-8 (IL-8) and fecal calprotectin levels were determined.RESULTS:Small intestinal HD-5 and TCF-4 mRNA were significantly reduced in pediatric ileal CD (L1+L3) (P=0.022 and P=0.0005, respectively) and were significantly correlated (r=0.499; P=0.0001). In ileal but not colonic CD, TCF-4 was also reduced in the colon (P=0.005). Importantly, both HD-5 and TCF-4 were independent of inflammation, as measured by IL-8 expression or fecal calprotectin. In contrast to the small intestine, colonic Paneth cell HD-5 mRNA was significantly elevated in colonic CD (L2) (P=0.026) and was correlated with fecal calprotectin levels (r=0.481; P=0.020).CONCLUSIONS:In this study, we describe a specific decrease in HD-5 and TCF-4 mRNA expression levels in children with ileal CD. In the small intestine, this decrease was independent of current inflammation, whereas inflammation seems to induce Paneth cell metaplasia in the colon. Our data extend the hypothesis of an important role of antimicrobial host defense in pediatric CD patients.

A 3' UTR transition within DEFB1 is associated with chronic and aggressive periodontitis.
Genes Immun. 2010 Jan;11(1):45-54.
Periodontal diseases are complex inflammatory diseases and affect up to 20% of the worldwide population. An unbalanced reaction of the immune system toward microbial pathogens is considered as the key factor in the development of periodontitis. Defensins have a strong antimicrobial function and are important contributors of the immune system toward maintaining health. Here, we present the first systematic association study of DEFB1. Using a haplotype-tagging single nucleotide polymorphism (SNP) approach, including described promoter SNPs of DEFB1, we investigated the associations of the selected variants in a large population (N=1337 cases and 2887 ethnically matched controls). The 3' untranslated region SNP, rs1047031, showed the most significant association signal for homozygous carriers of the rare A allele (P=0.002) with an increased genetic risk of 1.3 (95% confidence interval: 1.11-1.57). The association was consistent with the specific periodontitis forms: chronic periodontitis (odds ratio=2.2 (95% confidence interval: 1.16-4.35), P=0.02), and aggressive periodontitis (odds ratio=1.3 (95% confidence interval 1.04-1.68), P=0.02). Sequencing of regulatory and exonic regions of DEFB1 identified no other associated variant, pointing toward rs1047031 as likely being the causative variant. Prediction of microRNA targets identified a potential microRNA-binding site at the position of rs1047031.

Molecular diagnostics of psoriasis, atopic dermatitis, allergic contact dermatitis and irritant contact dermatitis.
Br J Dermatol. 2010 Mar;162(3):568-78.
ABSTRACT Background: Microarray studies on the epidermal transcriptome in psoriasis and atopic dermatitis have revealed genes with disease-specific expression in keratinocytes of lesional epidermis. These genes are possible candidates for disease-specific pathogenetic changes, but could also provide a tool for molecular diagnostics of inflammatory skin conditions in general. Objectives: To analyze if gene expression signatures as found in purified epidermal cells from atopic dermatitis are also present in other eczematous conditions such as allergic and irritant contact dermatitis. Methods: We used real-time quantitative PCR, immunohistochemistry and bioinformatics to investigate gene expression in different forms of eczema. Normal epidermis and psoriatic epidermis were analyzed for comparison. Results: Carbonic anhydrase II was highly induced in epidermis from all forms of eczema but not in psoriasis. Remarkably, the presumed neuron-specific Nel-like protein 2 showed a strong induction only in atopic dermatitis epidermis. Interleukin-1F9, elafin, beta-defensin-2 and vanin-3 were strongly induced in psoriasis, but not in any type of eczema. High levels of the chemokines CCL17 and CXCL10 were predominantly found in epidermis of allergic contact dermatitis. The chemokine CXCL8 was highly expressed in psoriasis, atopic dermatitis, and allergic contact dermatitis but not in irritant contact dermatitis. Cluster analysis or multinomial logistic regression indicated that expression levels of a set of seven genes are a strong predictor of the type of inflammatory response. Conclusions: These observations contribute to molecular diagnostic criteria for inflammatory skin conditions.

Functional polymorphisms of DEFB1 gene in type 1 diabetes Brazilian children.
Autoimmunity. 2009 Aug;42(5):406-13.
We analyzed three functional 5' un-translated region beta-defensin 1 (DEFB1) single nucleotide polymorphism (SNPs) in a group of 170 type 1 diabetes (T1D) patients. In order to evaluate the SNPs influence on the disease onset and the development of other autoimmune disorder, such as celiac disease (CD) and autoimmune thyroid disease (AITD), patients were stratified according to the presence of AITD, CD, and both AITD and CD. As control group, we studied 191 healthy children and adolescent not presenting a familiar historic of T1D, CD or AITD. DEFB1 SNPs were in Hardy-Weinberg equilibrium both in healthy controls and T1D patients, as well in the T1D patients stratified according to the presence of other autoimmune disorder(s). Allele, genotype, and haplotype frequencies of T1D patients globally considered were comparable to healthy controls ones. No evidence of any association of DEFB1 SNPs with the onset of AIDT, CD, and both AITD and CD on T1D patients was evidenced. Only a minor trend was found for an increased frequency of the - 20 G allele in T1D patients only presenting AITD vs. T1D patients not presenting AITD or CD, as well as an increase of those haplotypes comprising the - 20 G allele when compared with the GCA haplotype. We also evaluated the influence of functional DEFB1 SNPs on the age of T1D onset: no significant statistical conclusion was achieved. Further studies are envisaged, in order to elucidate the possible role of functional DEFB1 polymorphisms in the onset of TD1 and other autoimmune-related disorders.

Association of higher DEFB4 genomic copy number with Crohn's disease.
Am J Gastroenterol. 2010 Feb;105(2):354-9.
OBJECTIVES:Human beta-defensin 2 (hBD-2 or DEFB4) is a highly inducible, antimicrobial peptide, which may have an important role in the innate immune response at epithelial surfaces. Genomic copy number of DEFB4 is polymorphic, with most individuals possessing 3-5 copies. Increased DEFB4 copy number is a susceptibility factor for psoriasis, whereas a single study in a Crohn's disease (CD) cohort reported that decreased DEFB4 copy number is associated with colonic inflammation. Here, we analyze association of DEFB4 copy number with CD in a New Zealand case-control cohort of European origin.METHODS:DEFB4 gene copy number was determined using TaqMan quantitative PCR in 466 CD patients and 329 controls. DNA samples, independently genotyped for DEFB4 copy number by alternative methods, were used to validate the assay.RESULTS:Increased DEFB4 genomic copy number was seen in CD patients compared with controls. Individuals with >4 copies had a significantly higher risk of developing CD than those with <4 copies (odds ratio 1.54; 95% confidence interval 1.13-2.09, P=5e-05). DEFB4 genomic copy number did not differ by disease location within the CD cohort (P=0.948), nor did analysis of CD patients who had undergone surgery detect association of decreased DEFB4 genomic copy number (<4) in colonic CD compared with ileal CD (P=0.120).CONCLUSIONS:Our results indicate that elevated DEFB4 copy number is a risk factor for CD (irrespective of intestinal location), and challenge previous data supporting positive association of lower DEFB4 genomic copy number with colonic CD.

Localization of antimicrobial peptides human beta-defensins in minor salivary glands with Sjogren's syndrome.
Eur J Oral Sci. 2009 Oct;117(5):506-10.
Sjogren's syndrome is a common systemic autoimmune disease associated with inflammatory cells that infiltrate exocrine glands. The antimicrobial peptides human beta-defensin-1, human beta-defensin-2, and human beta-defensin-3 are expressed in various human epithelial cells and in normal salivary glands. Antimicrobial peptides provide local protection against infection and participate in inflammatory responses. Because of the presence of inflammation, we hypothesized that human beta-defensin expression in minor salivary glands may be increased in subjects with Sjögren's syndrome. However, the expression of human beta-defensins 1 and 2 was decreased in salivary glands affected by Sjogren's syndrome in comparison with the human beta-defensin expression patterns in salivary glands from normal subjects. In addition, the reduction in expression of human beta-defensin-2 was greater than the reduction in expression of human beta-defensin-1. The aforementioned result suggests that the reduction in expression of human beta-defensin-2 may occur earlier than the reduction in expression of human beta-defensin-1, which may lead to a greater decrease in human beta-defensin-2 than in human beta-defensin-1 during disease progression.