Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Inducible and constitutive beta-defensins are differentially expressed in Crohn's disease and ulcerative colitis.
Inflamm Bowel Dis. 2003 Jul;9(4):215-23.
Antimicrobial peptides such as defensins provide nonspecific mucosal defense against a multitude of microorganisms. Recently, it has been shown that luminal bacteria may invade the mucosa in inflammatory bowel diseases, suggesting a defect in innate mucosal immunity. The aim of this study was to investigate the expression of human beta-defensins (HBD) in controls, Crohn's disease (CD), ulcerative colitis (UC), and unspecific inflammation. Up to 4 biopsies were taken from 103 patients (33 controls, 24 with Crohn's disease, 36 with ulcerative colitis, 10 with unspecific colitis). Mucosal mRNA was measured using real-time fluorescence temperature cycler reverse-transcription polymerase chain reaction with primers for HBD-1, HBD-2, HBD-3, tumor necrosis factor alpha, and interleukin 8. Mucosal HBD-1 expression was marginally decreased in both CD and UC. HBD-2 was increased exclusively in UC but not in CD. The expression of the novel defensin HBD-3 was strongly correlated with HBD-2 and also raised predominantly in UC. The expression of both inducible beta-defensins was enhanced in the state of inflammation. Expression of HBD-2 showed a weak correlation with interleukin 8 only in inflamed CD biopsies but not with tumor necrosis factor alpha. The missing induction of both inducible beta-defensins in CD as compared with UC may cause a defect in barrier function that predisposes to bacterial invasion.

Hormonal contraception can suppress natural antimicrobial gene transcription in human endometrium.
Fertil Steril. 2003 Apr;79(4):856-63.
OBJECTIVE: To determine the effect of hormonal contraception with a combined oral contraceptive pill and levonorgestrel intrauterine system on the expression of the natural antimicrobials secretory leukocyte protease inhibitor, beta-defensins 1 and 2, and granulysin in human endometrium. DESIGN: Observational study. SETTING: Day case ward in a department of obstetrics and gynecology. PATIENT(S): Fifty seven women undergoing gynecologic procedures for benign conditions; 24 received no contraception for more than 3 months, 20 received a combined oral contraceptive for more than 3 months, and 13 wore a levonorgestrel intrauterine system for more than 3 months. MAIN OUTCOME MEASURE(S): Endometrial samples were collected from all women. Messenger RNA was extracted and quantitative polymerase chain reaction was used to investigate expression of secretory leukocyte protease inhibitor, beta-defensin 1, beta-defensin 2, and granulysin. Immunohistochemistry for secretory leukocyte protease inhibitor was performed. RESULT(S): All antimicrobials varied cyclically. The level of secretory leukocyte protease inhibitor was maximal in the late secretory and menstrual phase, beta-defensin 1 in the mid secretory phase, granulysin in the late secretory phase, and beta-defensin 2 in the menstrual phase. Use of a combined oral contraceptive or levonorgestrel intrauterine system use decreased messenger RNA expression of beta-defensin 1 and 2 and granulysin but not secretory leukocyte protease inhibitor. CONCLUSION(S): Endogenous and exogenous sex-steroid hormones, in the form of a combined oral contraceptive or levonorgestrel intrauterine system, influence gene transcription of secretory leukocyte protease inhibitor, beta-defensin 1, beta-defensin 2, and granulysin in the endometrium.

Bacterial vaginosis, vaginal fluid neutrophil defensins, and preterm birth.
Obstet Gynecol. 2003 May;101(5 Pt 1):862-8.
OBJECTIVE: To examine the association between bacterial vaginosis, vaginal fluid neutrophil defensins, and preterm birth. METHODS: Vaginal fluid specimens were obtained at 24-29 weeks' gestation from 242 cases with preterm birth and 507 noncases sampled using a case-cohort study design. We tested for bacterial vaginosis by Gram staining and Nugent scores and assayed for neutrophil defensins by enzyme-linked immunosorbent assay. Bacterial vaginosis was studied as a categoric variable (negative, intermediate, and positive), whereas defensins were studied as a continuous, categoric (based on percentiles), and dichotomous measure (presence versus absence). Three gestational age cut points were used to define preterm birth. Modified Cox proportional hazard models were used to evaluate the associations between bacterial vaginosis, defensins, and degree (less than 32, less than 34, and less than 37 weeks) and type (premature rupture of membranes, preterm labor) of preterm birth. RESULTS: Elevated vaginal fluid neutrophil defensins were not associated with birth before 37 weeks. Compared with women who did not have measurable vaginal fluid defensins, women with higher defensin levels (0-2.8 micro g/mL, 2.8-8.2 micro g/mL, and greater than 8.2 micro g/mL) had a greater risk of delivering before 32 weeks. Hazard ratios adjusted for maternal race and vaginal bleeding during pregnancy and 95% confidence intervals for these defensin levels were 1.7 (0.4, 6.9), 2.4 (0.7, 7.9), and 3.1 (1.0, 9.8), respectively. Bacterial vaginosis status did not influence the association between defensins and preterm birth. CONCLUSION: Elevated concentrations of vaginal fluid neutrophil defensins at 24-29 weeks' gestation might predict preterm birth before 32 weeks.

Increased concentrations of human beta-defensins in plasma and bronchoalveolar lavage fluid of patients with diffuse panbronchiolitis.
Thorax. 2003 May;58(5):425-30.
BACKGROUND: Human beta-defensin (HBD)-1 and -2 are antimicrobial peptides present in the respiratory tract. Recent reports have indicated reduced activity of beta-defensins in cystic fibrosis, suggesting that beta-defensins may play an important role in the pathological process of chronic respiratory tract infection. Diffuse panbronchiolitis (DPB) is a progressive disease characterised by frequent episodes of superimposed infection, typically caused by Pseudomonas aeruginosa. The aim of this study was to elucidate the role of these antimicrobial peptides in this disease. METHODS: The concentrations of HBD-1 and HBD-2 in plasma and bronchoalveolar lavage (BAL) fluid from 33 patients with DPB and 30 normal adults were measured by radioimmunoassay. Localisation of HBD-2 was investigated immunohistochemically in an open lung biopsy specimen obtained from a patient with DPB. RESULTS: High concentrations of HBD-1 and HBD-2 were noted in BAL fluid from DPB patients. Increased plasma concentrations of HBD-2, but not HBD-1, were found in patients with DPB compared with control subjects. In patients with DPB the HBD-2 concentration in BAL fluid correlated significantly with the numbers of cells recovered from the BAL fluid (total cells, neutrophils, and lymphocytes) and with the BAL fluid concentration of IL-1beta. Synthetic HBD-2, but not HBD-1, had dose dependent bactericidal activity against P aeruginosa. Treatment of 14 patients with macrolides significantly reduced BAL fluid concentrations of HBD-2 but not HBD-1 or plasma concentrations of HBD-1 and HBD-2. Immunohistochemistry of lung tissue showed localisation of HBD-2 in the epithelia of the distal bronchioles. CONCLUSIONS: These results indicate that beta-defensins, particularly HBD-2, participate in antimicrobial defence in the respiratory tract in DPB, and that the BAL fluid concentration of HBD-2 may be a useful marker of airway inflammation in patients with DPB.

Defensin pattern in chronic gastritis: HBD-2 is differentially expressed with respect to Helicobacter pylori status.
J Clin Pathol. 2003 May;56(5):352-7.
BACKGROUND/AIMS: Recent reports have suggested that Helicobacter pylori infection induces the mucosal antibiotic peptide human beta defensin 2 (HBD-2). Therefore, the present study investigated mRNA and peptide expression of four different defensins in the upper gastrointestinal tract in patients with H pylori positive and negative chronic gastritis. MATERIALS/METHODS: Biopsies from the oesophagus to the duodenum were taken during routine gastroscopy in 71 individuals. Total RNA was extracted and the reverse transcription polymerase chain reaction was performed with primers for human defensins 5 and 6 (HD-5/6) or HBD-1 and HBD-2. Paraffin wax embedded tissue from gastric resections was tested for HD-5, HBD-1, and HBD-2 by immunohistochemistry. RESULTS: Helicobacter pylori colonisation was associated with an increased percentage of positive biopsies with respect to HBD-2 in the corpus (p < 0.05). Helicobacter pylori had no impact on the gastric expression of HD-5 and HBD-1, whereas HD-6 was increased in the fundus. The abundant expression of alpha defensins in the duodenum and beta defensins in the oesophagus served as a positive control in each individual. Immunohistochemical analysis confirmed the presence of the HD-5, HBD-1, and HBD-2 peptides in gastric resection specimens. CONCLUSIONS: The recently described induction of HBD-2 upon H pylori infection was confirmed in a clinical setting of chronic gastritis. This phenomenon may be mediated by components of the pathogen itself or may occur secondary to immune events in chronic inflammation.

Antimicrobial peptides in amniotic fluid: defensins, calprotectin and bacterial/permeability-increasing protein in patients with microbial invasion of the amniotic cavity, intra-amniotic inflammation, preterm labor and premature rupture of membranes.
J Matern Fetal Neonatal Med. 2003 Jan;13(1):2-21.
OBJECTIVE: Neutrophil defensins (HNP 1-3), bactericidal/permeability-increasing protein (BPI) and calprotectin (MRP8/14) are antimicrobial peptides stored in leukocytes that act as effector molecules of the innate immune response. The purpose of this study was to determine whether parturition, premature rupture of the membranes (PROM) and microbial invasion of the amniotic cavity (MIAC) are associated with changes in amniotic fluid concentrations of these antimicrobial peptides. STUDY DESIGN: Amniotic fluid was retrieved by amniocentesis from 333 patients in the following groups: group 1, mid-trimester with a subsequent normal pregnancy outcome (n = 84); group 2, preterm labor and intact membranes without MIAC who delivered at term (n = 36), or prematurely (n = 52) and preterm labor with MIAC (n = 26); group 3, preterm PROM with (n = 26) and without (n = 26) MIAC; and group 4, term with intact membranes in the absence of MIAC, in labor (n = 52) and not in labor (n = 31). The concentrations of HNP 1-3, BPI and calprotectin in amniotic fluid were determined by specific and sensitive immunoassays. Placentae of patients in both preterm labor with intact membranes and preterm PROM groups who delivered within 72 h of amniocentesis were examined. Non-parametric statistics, receiver-operating characteristic (ROC) curves and Cox regression models were used for analysis. A p value of < 0.05 was considered statistically significant. RESULTS: Intra-amniotic infection was associated with a significant increase in amniotic fluid concentrations of immunoreactive HNP 1-3, BPI and calprotectin in both women with preterm labor and intact membranes, and women with preterm PROM. Preterm PROM was associated with a significant increase in amniotic fluid concentrations of immunoreactive HNP 1-3, BPI and calprotectin. Preterm parturition was associated with a significant increase in amniotic fluid concentrations of immunoreactive HNP 1-3, BPI and calprotectin, while parturition at term was associated with a significant increase in amniotic fluid concentrations of immunoreactive HNP 1-3. Among patients with preterm labor and intact membranes, elevation of amniotic fluid HNP 1-3, BPI and calprotectin concentrations was associated with intra-amniotic inflammation, histological chorioamnionitis and a shorter interval to delivery. CONCLUSION: MIAC, preterm parturition and preterm PROM are associated with increased amniotic fluid concentrations of immunoreactive HNP 1-3, BPI and calprotectin. Moreover, elevated amniotic fluid concentrations of BPI, immunoreactive HNP 1-3 and calprotectin are associated with intra-amniotic inflammation, histological chorioamnionitis and shorter amniocentesis-to-delivery interval in patients presenting with preterm labor with intact membranes.

Expression of beta-defensins in human middle ear cholesteatoma.
Acta Otolaryngol. 2003 Jan;123(2):236-40.
OBJECTIVE: To establish the eventual presence of human beta-defensins (hBD) in middle ea r cholesteatoma and paired retroauricular skin samples obtained during surgical treatment. MATERIAL AND METHODS: hBD were detected using immunohistochemical techniques and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: It is hypothesized that hBD-1 and -2 are upregulated in cholesteatoma in comparison with retroauricular skin. The immunohistochemical study demonstrated stronger hBD-2-positive staining in middle ear cholesteatoma in comparison with retroauricular skin. No significant differences in hBD-1 expression were detected between cholesteatoma and skin samples. RT-PCR established elevated expression of hBD-2 mRNA in middle ear cholesteatomas. HBD-1 mRNA was constitutively expressed in cholesteatoma and retroauricular skin specimens. CONCLUSION: These preliminary experimental results lead us to believe that hBD may play an important role in the chronic inflammatory state of middle ear cholesteatoma.

Cancer-specific loss of beta-defensin 1 in renal and prostatic carcinomas
Lab Invest. 2003 Apr;83(4):501-5
In a previous large-scale gene expression profiling study of renal epithelial neoplasms, human beta-defensin-1 (DEFB1) was found to be significantly down-regulated in conventional clear cell (renal) carcinoma. We have now completed an expanded expression analysis of this gene. We performed immunohistochemical analysis for the DEFB1 protein in clinical specimens of both renal cell carcinoma and prostate cancer. In a subset of prostate cancers, we performed laser capture microdissection and RT-PCR to correlate mRNA levels with protein levels. Overall, 82% of prostate cancers exhibit either complete loss of protein expression or only minimal expression, whereas the adjacent benign epithelium retained expression in all cases. Similarly, 90% of renal cell carcinomas show cancer-specific loss of DEFB1 protein. In the prostate cancer subset analysis, mRNA levels correlate with protein levels. We have thus demonstrated the cancer-specific down-regulation of DEFB1 in a large sample of prostatic and renal carcinomas and validated one of the key findings of previous cancer gene profiling studies of prostatic and renal neoplasia.

Antimicrobial polypeptides of human vernix caseosa and amniotic fluid: implications for newborn innate defense.
Pediatr Res. 2003 Feb;53(2):211-6.
Antimicrobial peptides/proteins are widespread in nature and play a critical role in host defense. To investigate whether these components contribute to surface protection of newborns at birth, we have characterized antimicrobial polypeptides in vernix caseosa (vernix) and amniotic fluid (AF). Concentrated peptide/protein extracts were obtained from 11 samples of vernix and six samples of AF and analyzed for antimicrobial activity using an inhibition zone assay. Proteins/peptides in all vernix extracts exhibited strong antibacterial activity against Bacillus megaterium (strain Bm11), in addition to antifungal activity against Candida albicans, whereas AF-derived proteins/peptides showed only the former activity. Fractions obtained after separation by reverse-phase HPLC exhibited antibacterial activity, with the most pronounced activity in a fraction containing alpha-defensins (HNP1-3). The presence of HNP1-3 was proved by dot blot analysis and confirmed by mass spectrometry. Lysozyme and ubiquitin were identified by sequence analysis in two fractions with antibacterial activity. Fractions of vernix and AF were also positive for LL-37 with dot blot and Western blot analyses, and one fraction apparently contained an extended form of LL-37. Interestingly, psoriasin, a calcium-binding protein that is up-regulated in psoriatic skin and was found recently to exhibit antimicrobial activity, was characterized in the vernix extract. The presence of all of these antimicrobial polypeptides in vernix suggests that they are important for surface defense and may have an active biologic role against microbial invasion at birth.

Single-nucleotide polymorphisms and haplotype analysis in beta-defensin genes in different ethnic populations.
Genet Test. 2002 Winter;6(4):261-9.
Beta-defensins are cationic antimicrobial peptides expressed by epithelial cells and exhibit antibacterial, antifungal, and antiviral properties. The defensins are part of the innate host defense network and may have a significant protective role in the oral cavity and other mucosa. Defects or alteration in expression of the beta-defensins may be associated with susceptibility to infection and mucosal disorders. We examined the occurrence of single-nucleotide polymorphisms (SNPs) in the human beta-defensin genes DEFB1 and DEFB2 encoding human beta-defensin-1 and -2 (hBD-1, hBD-2), respectively, in five ethnic populations and defined haplotypes in these populations. Fifteen SNPs were identified in both DEFB1 and DEFB2. Coding region SNPs were found in very low frequency in both genes. One nonsynonymous DEFB1 SNP, G1654A (Val --> Ile), and one nonsynonymous DEFB2 SNP, T2312A (Leu --> His), were identified. Seven sites in each gene exhibited statistically significant differences in frequency between ethnic groups, with the greatest variation in the promoter and in the 5'-untranslated region of DEFB1. DEFB1 displayed 10 common haplotypes, including one cosmopolitan haplotype. Eight common haplotypes were found in DEFB2, including one cosmopolitan haplotype shared among all five ethnic groups. Our results show that genotypic variability among ethnic groups will need to be addressed when performing associative genetic studies of innate defense mechanisms and susceptibility to disease.