Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Midpregnancy vaginal fluid defensins, bacterial vaginosis, and risk of preterm delivery
Obstet Gynecol. 2008 Sep;112(3):524-31
OBJECTIVE: To assess relations among midpregnancy vaginal defensin levels, a component of the host innate immune response, bacterial vaginosis, and risk of preterm delivery. These relations are compared across race groups because previous studies have repeatedly shown that the prevalence of bacterial vaginosis and the risk of preterm delivery are greater in African-American women compared with that in white women. METHODS: Data are from a prospective study that enrolled pregnant women from 52 clinics in five Michigan communities. In the study subcohort, defensins (human neutrophil peptides 1, 2 and 3) and bacterial vaginosis (Nugent criteria) were measured in vaginal fluid collected at enrollment (15th through 27th week of pregnancy) from 1,031 non-Hispanic white and African-American women (787 term, 244 preterm). Preterm deliveries were categorized by clinical circumstances, ie, spontaneous and medically indicated. RESULTS: Among African Americans, vaginal human neutrophil peptides 1-3 levels greater than or equal to the median were associated with bacterial vaginosis and specifically with spontaneous preterm delivery only (adjusted odds ratio 2.3, 95% confidence interval 1.2-4.3). Once African-American women were stratified by human neutrophil peptide 1-3 levels, bacterial vaginosis added nothing to the prediction of spontaneous preterm delivery risk. None of the above associations were observed in non-Hispanic whites. CONCLUSION: The relations among human neutrophil peptide 1-3 levels, bacterial vaginosis, and preterm delivery vary by race group. In African Americans, midpregnancy human neutrophil peptide 1-3 levels were more informative to preterm delivery risk than was bacterial vaginosis, suggesting an important role for host response. In addition, elevated human neutrophil peptide 1-3 levels may be a marker for particular high-risk vaginal milieus that are not distinguished by the current bacterial vaginosis Nugent scoring system.

Analysis of neutrophil-derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL-37 in the innate immune response against periodontogenic bacteria
Oral Microbiol Immunol. 2008 Aug;23(4):328-35
INTRODUCTION: During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil-derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. METHODS: GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real-time polymerase chain reactions. The amounts of myeloperoxidase and alpha-defensins (HNP1-3) were determined by enzyme-linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL-37) was assayed by Western blot. RESULTS: Myeloperoxidase concentration was not correlated with levels of LL-37 and HNP1-3 in samples from patients, compared to controls. The amount of HNP1-3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL-37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. CONCLUSION: The lack of a correlation between LL-37, HNP1-3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL-37, because the 11-kDa cathelicidin-derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL-37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.

Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis.
Gut. 2008 Oct;57(10):1398-405
OBJECTIVE: To investigate differential intestinal gene expression in patients with ulcerative colitis and in controls. DESIGN: Genome-wide expression study (41,058 expression sequence tags, 215 biopsies). SETTING: Western General Hospital, Edinburgh, UK, and Genentech, San Francisco, USA. PATIENTS: 67 patients with ulcerative colitis and 31 control subjects (23 normal subjects and 8 patients with inflamed non-inflammatory bowel disease biopsies). INTERVENTIONS: Paired endoscopic biopsies were taken from 5 specific anatomical locations for RNA extraction and histology. The Agilent microarray platform was used and confirmation of results was undertaken by real time polymerase chain reaction and immunohistochemistry. RESULTS: In healthy control biopsies, cluster analysis showed differences in gene expression between the right and left colon. (chi(2) = 25.1, p<0.0001). Developmental genes, homeobox protein A13 (HOXA13), (p = 2.3x10(-16)), HOXB13 (p<1x10(-45)), glioma-associated oncogene 1 (GLI1) (p = 4.0x10(-24)), and GLI3 (p = 2.1x10(-28)) primarily drove this separation. When all ulcerative colitis biopsies and control biopsies were compared, 143 sequences had a fold change of >1.5 in the ulcerative colitis biopsies (0.01>p>10(-45)) and 54 sequences had a fold change of <-1.5 (0.01>p>10(-20)). Differentially upregulated genes in ulcerative colitis included serum amyloid A1 (SAA1) (p<10(-45)) the alpha defensins 5 and 6 (DEFA5 and 6) (p = 0.00003 and p = 6.95x10(-7), respectively), matrix metalloproteinase 3 (MMP3) (p = 5.6x10(-10)) and MMP7 (p = 2.3x10(-7)). Increased DEFA5 and 6 expression was further characterised to Paneth cell metaplasia by immunohistochemistry and in situ hybridisation. Sub-analysis of the inflammatory bowel disease 2 (IBD2) and IBD5 loci, and the ATP-binding cassette (ABC) transporter genes revealed a number of differentially regulated genes in the ulcerative colitis biopsies. CONCLUSIONS: Key findings are the expression gradient in the healthy adult colon and the involvement of novel gene families, as well as established candidate genes in the pathogenesis of ulcerative colitis.

Microanalysis of an antimicrobial peptide, beta-defensin-2, in the stratum corneum from patients with atopic dermatitis.
Br J Dermatol. 2008 Jul;159(1):97-104.
BACKGROUND: Antimicrobial peptides, such as defensin and cathelicidin, have recently been reported to play important roles in host defence and in cutaneous innate immunity. Although beta-defensin-2 has been reported to be downregulated in the skin of patients with atopic dermatitis (AD), little is known about its role in the colonization of Staphylococcus aureus in the stratum corneum of patients with AD. A precise evaluation of these peptides in the stratum corneum as an antimicrobial barrier against S. aureus colonization has not yet been performed. OBJECTIVES: To compare beta-defensin-2 levels in the skin of patients with AD and healthy controls. METHODS: We developed a microanalytical technique to measure beta-defensin-2 in the stratum corneum using a combination of immunoprecipitation and Western blotting. RESULTS: beta-Defensin-2 in the stratum corneum was significantly higher in AD lesional skin compared with healthy control skin. The beta-defensin-2 content in AD lesional skin also increased in proportion to the severity of the disease. Counting bacterial colonies revealed higher populations of S. aureus on lesional and nonlesional skin surfaces of patients with AD compared with healthy controls. Comparison of S. aureus colony numbers and beta-defensin-2 levels demonstrated a positive correlation (r = 0.342, P = 0.004, n = 67) between both factors. CONCLUSIONS: Collectively, these findings suggest that beta-defensin-2 is induced in response to bacteria, injury or inflammatory stimuli and is not associated with vulnerability to S. aureus colonization in the skin of patients with AD.

Increased levels of human neutrophil alpha-defensins in chronic venous leg ulcers
J Dermatol Sci. 2008 Aug;51(2):131-4
Here we investigated the occurrence and potential antimicrobial activity of defensins in wound fluid as well as tissues of chronic venous ulcers. 15 patients (8 women, 7 men, mean age of 80 years) with CVLU were included. We detected significantly increased levels of alpha-defensins in CVLU fluid when compared with AW fluid. In contrast to normal skin, both chronic and acute wounds were characterized by an inflammatory infiltrate. IH of chronic ulcer tissues identified alpha-defensins in the leukocyte infiltrate, compatible with the fact that PMNs contain alpha-defensins. Defensins were also found peri/extracellularly, likely reflecting PMN degranulation and release of defensins.

Differential mRNA expression of antimicrobial peptides and proteins in atopic dermatitis as compared to psoriasis vulgaris and healthy skin
Int Arch Allergy Immunol. 2008;147(1):17-24
BACKGROUND: Patients with atopic dermatitis (AD) are prone to have skin infections. We aimed to investigate mRNA expression levels of various antimicrobial peptides and proteins (AMPs) in AD patients, and compare it with psoriasis vulgaris (PV) patients and healthy subjects. METHODS: Skin biopsies were obtained from healthy subjects and patients with AD and PV. Quantitative real-time RT-PCR was used to determine the mRNA levels of human beta-defensin (hBD)-1, hBD-2, hBD-3, LL-37, psoriasin, RNase 7, interferon-gamma, and interleukin-10 (IL-10). RESULTS: Except for LL-37, mRNA of hBDs, psoriasin, and RNase 7 was significantly higher expressed in AD (n = 42) and/or PV (n = 35) patients when compared to controls (n = 18). While PV lesions showed significantly higher mRNA hBD-2 levels than lesions of AD, the latter was associated with significantly higher mRNA levels of RNase 7 when compared to PV. A significant positive correlation of hBD expression was observed both in AD patients and PV patients. hBD mRNA levels of AD skin correlated with psoriasin and RNase 7 levels. hBD-1 mRNA expression correlated with AD activity and IL-10 mRNA expression. CONCLUSIONS: Most AMPs investigated in this study proved to be overexpressed in AD as well as PV when compared to controls. However, a statistically significant difference in AMP mRNA expression between AD and PV was only found for hBD-2 and RNase 7. A moderate-to-strong linear relationship between the mRNA expression of particular AMPs appears to exist in AD, and to a lesser extent in PV as well.

Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells.
Am J Rhinol. 2008 Mar-Apr;22(2):115-21
BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNPs) is a disorder characterized by persistent eosinophilic Th2 inflammation and frequent sinonasal microbial colonization. It has been postulated that an abnormal mucosal immune response underlies disease pathogenesis. The relationship between Th2 inflammatory cytokines and the innate immune function of sinonasal epithelial cells (SNECs) has not been explored. METHODS: Human SNECs (HSNECs) isolated from control subjects and patients with CRS were assessed for expression of antimicrobial innate immune genes and proinflammatory cytokine genes by real-time polymerase chain reaction, ELISA, and flow cytometry. A model of the Th2 inflammatory environment was created by exposure of primary HSNEC to the Th2 cytokine interleukin (IL)-4 or IL-13 for 36 hours, with subsequent assessment of innate immune gene expression. RESULTS: HSNEC obtained from CRSwNP patients displayed decreased expression of multiple antimicrobial innate immune markers, including toll-like receptor 9, human beta-defensin 2, and surfactant protein A. Baseline expression of these genes by normal and CRS HSNEC in culture is significantly down-regulated after incubation with IL-4 or IL-13. CONCLUSION: Expression of multiple innate immune genes by HSNEC is reduced in CRSwNP. One mechanism appears to be a direct effect of the leukocyte-derived Th2 cytokines present in the sinonasal mucosa in CRSwNP. Impaired mucosal innate immunity may contribute to microbial colonization and abnormal immune responses associated with CRSwNP.

Interaction between seroreactivity to microbial antigens and genetics in Crohn's disease: is there a role for defensins?
Tissue Antigens. 2008 Jun;71(6):552-9
Antibodies against different microbial epitopes are associated with disease phenotype, may be of diagnostic importance and may reflect a loss of tolerance in Crohn's disease (CD). Recently, an association was reported between the presence of these antibodies and mutations in pattern receptor genes. Our aim was to investigate whether mutations in various genes other than NOD2/CARD15 or TLR4 associated with CD (NOD1/CARD4, DLG5 and DEFB1) may influence the presence of antibodies against bacterial proteins and carbohydrates in a Hungarian cohort of CD patients. Three hundred and seventy-six well-characterized, unrelated, consecutive CD patients (male/female: 191/185, age at onset: 29.1 +/- 12.9 years, duration: 7.9 +/- 11.7 years) were investigated. Sera were assayed for anti-Omp, anti-Saccharomyces cerevisiae antibodies (ASCAs) immunoglobulin (Ig) A and IgG, and antibodies against a mannan epitope of S. cerevisiae (gASCA), laminaribioside (ALCA), chitobioside (ACCA), and mannobioside (AMCA). NOD1/CARD4, DLG5 and DEFB1 variants were tested by polymerase chain reaction-restriction fragment length polymorphism, and DEFB1 was genotyped in a subgroup of 160 patients. Detailed clinical phenotypes were determined by reviewing the patients' medical charts. The carriage of DEFB1 20A variant alleles less frequently led to antiglycan positivity compared with patients without (29.6% vs 46.2%, OR: 0.49, 95% CI: 0.25-0.97), regardless of disease location or behavior. Similar tendency was observed for DEFB1 44G (present: 21.6% vs absent: 10.2%, P = 0.06) and ALCA. A gene or serology dosage effect was not observed. However, no association was found between the DEFB1 G52A, DLG5 R30Q, and NOD1/CARD4 E266K variants and any of the serology markers. We found that variants in human beta-defensin 1 gene are inversely associated with antiglycan antibodies, further confirming an important role for innate immunity in the pathogenesis of CD.

Single nucleotide polymorphisms in human Paneth cell defensin A5 may confer susceptibility to inflammatory bowel disease in a New Zealand Caucasian population.
Dig Liver Dis. 2008 Sep;40(9):723-30
BACKGROUND: Human Paneth cell alpha-defensins, especially DEFA5, are involved in maintaining homeostasis of the human microbial microflora. Since breakdown of normal mucosal antibacterial defence occurs in inflammatory bowel disease (IBD), variants in the DEFA5 gene could be associated with IBD risk. SUBJECTS: A cohort of 25 patients with indeterminate colitis (IC), 405 with ulcerative colitis (UC), and 385 with Crohn's disease (CD), were compared with 201 control individuals from the Canterbury region in New Zealand. METHODS: A 15 kb haplotype block surrounding DEFA5 contained 35 HapMap markers which were polymorphic in Caucasians. Four markers (A-D) were selected to tag 27 of the 35 markers at r(2)>0.68, and were genotyped in DNA samples. RESULTS: Minor allele frequencies for all single nucleotide polymorphisms (SNPs) were somewhat elevated in patients. Subgroup analysis showed SNP A had odds ratio 1.44 in UC patients with pancolitis (95% C.I. 1.07-1.94), SNP B odds ratio 2.37 in CD patients with onset prior to 17 years age (95% C.I. 1.12-5.03), SNP C odds ratio 1.68 in UC patients with left colonic localisation (95% C.I. 1.12-2.52), and SNP D had odds ratio 1.56 in CD patients with one or more relatives with IBD (95% C.I. 1.03-2.35). Two two-marker haplotypes and one three-marker haplotype were associated with UC (p-values 0.025-0.05). CONCLUSIONS: The SNPs genotyped in our study were surrogates for common variants, and observed associations between these and IBD status are likely due to linkage disequilibrium with a functional common DEFA5 variant. Identifying such functional variants will be prioritized in subsequent work.

Tumor necrosis factor-alpha, Interleukin-10, and alpha-defensins in plasma and breast milk of HIV-infected highly active antiretroviral therapy-treated and untreated pregnant women in Mozambique
J Acquir Immune Defic Syndr. 2008 Apr 15;47(5):647-9