Clinical Studies

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Last updated: 13th August 2010

Clinical Studies
Human beta-Defensins in different states of diseases of the tonsilla palatina.
Eur Arch Otorhinolaryngol. 2010 May;267(5):821-30.
Tonsils are believed to play an important role during the development of the immune system. Although diseases of the tonsils like hypertrophy of the tonsil, acute tonsillitis, chronic tonsillitis or peritonsillar abscess are common, little is known about the underlying pathophysiology. Little is known about antimicrobial peptides produced by the tonsils. The human beta-Defensins 1-3 (hBD1-3) are naturally produced "antibiotics" with antimicrobial activity against different bacteria, fungi, and viruses. The objective of the study was to determine the concentrations for hBD1-3 in different states of diseases of the tonsilla palatina. After tonsillectomy and tissue fixation in formalin, total proteins were isolated from 38 samples (11 hypertrophy of the tonsil, 8 acute tonsillitis, 11 chronic tonsillitis, 8 peritonsillar abscesses). The protein concentration was determined and ELISA for hBD1-3 were performed. We also conducted immunofluorescence double stainings for the co-expression of streptococcus group A and hBD1-3. We could verify a significant difference for the mean hBD1 score of the acute tonsillitis in comparison to the hyperplastic tonsil, the chronic tonsillitis, and the peritonsillar abscess. There was no statistically significant difference in the hBD2 and hBD3 concentrations between the four groups. The immunofluorescence stainings showed that hBD1-3 and the streptococcus group A in the same place. We conclude that in the hyperplastic tonsilla palatina hBD1-3 play an important role. The mouth is constantly faced with a high bacterial load. During a tonsillitis, the hBD1 concentration is lower than in the non-acute infected tonsil because hBD1 is being consumed for fighting the bacterial infection. But, the existence of hBD1-3 in the tonsil cannot prevent the tonsillitis to become chronic.

Study of human neutrophil peptides in saliva by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Rapid Commun Mass Spectrom. 2009 Oct;23(19):3220-6.
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is used to rapidly characterize the human neutrophil peptides - HNP 1, 2, and 3 - in saliva. The saliva excreted from the parotid and sublingual/submandibular glands of 70 individuals were collected and examined using MALDI-TOF. The MALDI approach requires no sample pretreatment other than mixing the saliva-absorbing material with the matrix and drying under ambient conditions. Tissue paper was the best material for collecting the saliva samples because of its strong texture and high absorbance, and sinapinic acid was the best MALDI matrix for the analysis of the HNPs. HNPs were detected in almost all the samples collected from the parotid glands, with no obvious differences among age or gender. In contrast, the distribution of the HNPs in the samples collected from the sublingual/submandibular glands was age-dependent: no HNPs were detected for those collected from individuals younger than 30, but the HNPs were present in all of the samples collected from those older than 60 years. The increased probability of detecting saliva HNPs with age suggests that HNPs may function as a biomarker for aging.

beta-Defensin 1 haplotype associated with postoperative endophthalmitis.
Acta Ophthalmol. 2009 Aug 25. [Epub ahead of print]
Abstract. Purpose: Endophthalmitis is a rare but sight-threatening complication of intraocular surgery. beta-Defensins are antimicrobial peptides that appear to be important components of the ocular immune response. We propose that variation in defensin genes may alter susceptibility to endophthalmitis. Methods: Post-cataract endophthalmitis patients (n = 28) and post-cataract controls (n = 75) were recruited and DNA samples extracted. The beta-defensin 1 gene (DEFB1) was screened for single-nucleotide polymorphisms (SNPs) using bidirectional sequencing. Case-control statistical assessment was undertaken for both the individual polymorphic loci observed and combined haplotypes using PHASE software. Results: We identified 19 SNPs and observed strong linkage disequilibrium within the gene. We found that the three-SNP haplotype -688C/-44C/-20A was associated strongly with endophthalmitis [odds ratio (OR) = 8.88 (1.74, 45.42), corrected p = 0.0095]. Furthermore, we uncovered several trends, including increased prevalence of the -44CC genotype in the endophthalmitis group. Conclusion: We have shown previously that the -44CC SNP genotype was present in a single case of bilateral endophthalmitis. In this study, we found this genotype to be more common in the endophthalmitis group and a mini-haplotype including this SNP was associated strongly with endophthalmitis. There is functional evidence that this genetic profile decreases transcription of the beta-defensin 1 peptide and could therefore reduce the innate ocular immune defence.

Loss of human beta-defensin 1, 2, and 3 expression in oral squamous cell carcinoma.
Oral Microbiol Immunol. 2009 Oct;24(5):353-60.
INTRODUCTION: Human beta-defensins (HBDs) are cationic, antimicrobial peptides produced by epithelial cells and involved in various aspects of the innate and acquired immune responses. They are expressed by oral tissues as constitutive and inducible genes. Recently, single nucleotide polymorphisms (SNPs) of beta-defensins have been correlated with increased susceptibility to certain diseases. Studies have reported altered expression of beta-defensins in cancers suggesting their involvement in carcinogenesis. The purpose of this study was to evaluate the regulation of HBD-1 (also published as DEFB1), HBD-2 (DEFB4) and HBD-3 (DEFB103A) ( and HBD-1 SNPs in oral squamous cell carcinoma cell lines (OSCC) and healthy gingival keratinocytes. METHODS: beta-defensin expression was quantitatively assessed using real-time polymerase chain reactions in OSCC and control cell lines after exposure to interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma. Control data were obtained in a previous study. DNA from 19 OSCC cell lines and 44 control subjects were extracted and the HBD-1 region spanning the 5' untranslated region to the first intron was sequenced and analysed for SNP identification and distribution. RESULTS: HBD-1 and HBD-2 basal messenger RNA expression were significantly lower in OSCC. In addition, the ability to be induced was significantly reduced in OSCC for all three beta-defensins. Four HBD-1 SNPs were differentially distributed between cancer and control populations. Genotype distribution at the HBD-1 locus also suggested loss of heterozygosity in OSCC. CONCLUSIONS: The genetic variation observed in OSCC compared with that in control cell lines may account for differences in beta-defensin expression. These results suggest a putative role for beta-defensins in carcinogenesis and indicate that beta-defensins may be useful markers of OSCC.

The role of human beta defensins 2 and 3 in the second trimester amniotic fluid in predicting preterm labor and premature rupture of membranes.
Arch Gynecol Obstet. 2010 May;281(5):793-9.
AIM: Human beta defensins 2 (HBD2) and 3 (HBD3) are peptides expressed in the amnion and chorion. This is a matched case control study conducted in our Department to determine whether second trimester amniotic fluid HBD2 and HBD3 concentrations measured at the time of genetic amniocentesis could be potential markers of preterm labor prediction. METHODS: Amniotic fluid HBD2 and HBD3 were determined by an enzyme-linked immunosorbent assay (ELISA) Women with preterm labor were defined as cases (N = 41) while for each case a woman matched for age delivering at term served as control (N = 41). Subgroup analysis was conducted to examine possible associations of HBD2 and HBD3 in cases of premature rupture of membranes. Nineteen women with preterm labor and premature rupture of membranes were defined as cases while for every case a woman matched for maternal age delivering at term served as control (N1 = 19). Results were presented as odds ratios (OR) and 95% confidence intervals. Statistical analysis used STATA 8.2 and SPSS 11.5 edition. A P-value of <0.05 was considered statistically significant. RESULTS: Amniotic fluid concentrations of HBD2 at the time of genetic amniocentesis were positively associated with preterm premature rupture of membranes (P = 0.028), but not with preterm labour. No association of HBD3 and preterm birth was documented. CONCLUSION: Second trimester amniotic fluid HBD2 might be a predictor of premature rupture of membranes.

Identification of macrophage migration inhibitory factor and human neutrophil peptides 1-3 as potential biomarkers for gastric cancer.
Br J Cancer. 2009 Jul 21;101(2):295-302.
BACKGROUND: Proteomic methods have the potential to meet the urgent need for better cancer biomarkers. We have used a range of proteomic analyses of serum and tissue from gastric cancer patients and relevant controls to discover biomarkers for gastric cancer. METHODS: Surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI) and antibody arrays were used to compare protein expression in 21 pairs of gastric cancer tissue and adjacent normal mucosa and serum from 51 gastric cancer patients and 29 patients with benign gastric diseases. Expression differences were confirmed by enzyme-linked immunosorbent assay. RESULTS: Tissue analysis shows human neutrophil peptides 1-3 (HNPs 1-3) elevated 10-fold (P=0.001) in gastric cancer relative to adjacent normal mucosa. Macrophage migration inhibitory factor (MIF) was increased five-fold (P=1.84 x 10(-7)) in the serum of gastric cancer patients relative to individuals with benign gastric disease. The large increase in MIF concentration in serum gives an area under the receiver operating characteristic curve of 0.85. CONCLUSIONS: Proteomic analyses of serum and tissue indicate that HNPs 1-3 and MIF have potential as biomarkers for gastric cancer. In particular MIF may be useful, either alone or in combination with other markers, for diagnosing and monitoring gastric cancer.

Influence of standard treatment on ileal and colonic antimicrobial defensin expression in active Crohn's disease.
Aliment Pharmacol Ther. 2009 Sep 15;30(6):621-33.
Background: Current therapy of Crohn's Disease (CD), a chronic intestinal inflammation, is primarily directed against inflammatory responses. In pathophysiology, recent findings suggest a central role of the innate immune barrier. Important factors providing this barrier are antimicrobial peptides like the alpha- and beta-Defensins. Little is known about in vivo effects of common drugs on their expression. Aim: We analysed the influence of corticosteroids, azathioprine and aminosalicylate treatment on ileal and colonic antimicrobial peptides in active Crohn's Disease and also assessed the role of inflammation. Methods: We measured the expression of antimicrobial peptides and pro-inflammatory cytokines in 75 patients with active CD. Results: Ileal and colonic alpha- and beta-defensins as well as LL37 were unaffected by corticosteroids, azathioprine or aminosalicylate treatment. Additionally we did not observe a negative coherency between Paneth cell alpha-defensins and any measured cytokines. HBD2 and LL37 unlike HBD1 levels were linked to inflammatory cytokines and increased in highly inflamed samples. Conclusion: Current oral drug treatment seems to have no major effect on the expression of antimicrobial peptides. In contrast to HBD2 and LL37, Ileal levels of HD5 and HD6 and colonic HBD1 level are independent of current inflammation. Further drugs should aim to strengthen protective innate immunity.

Role of beta-defensin-1 polymorphisms in mother-to-child transmission of HIV-1.
J Acquir Immune Defic Syndr. 2009 May 1;51(1):13-9.
BACKGROUND AND OBJECTIVES: Mother-to-child transmission (MTCT) of HIV-1, the main source of pediatric AIDS, is multifactorial. Defensins provide microbial barriers and function as effectors of innate immunity. This study investigated the relationship between genetic variants of beta-defensin-1 gene and MTCT of HIV-1. PATIENTS AND METHODS: Three hundred children, 118 HIV-1 infected and 182 HIV-1 uninfected, born to HIV-1-infected mothers who had not undergone antiretroviral therapy during pregnancy, and 84 HIV-1-infected mothers were analyzed. The single nucleotide polymorphisms -44C/G (rs1800972) and -52G/A (rs1799946) were genotyped by TaqMan allelic discrimination assay and sequencing. Statistical analyses were performed using SNPStats and Bonferroni correction for multiple tests. RESULTS: In children, the -52GG genotype and the -44G/-52G haplotype had a protective role against HIV-1 infection [odds ratio (OR) = 0.52, 95% confidence interval (CI) 0.31 to 0.86, P = 0.03 and OR = 0.50, 95% CI 0.31 to 0.83, P = 0.014, respectively]. In mothers, the -52GG genotype and the -44G/-52G haplotype were associated with low levels of HIV-1 plasma viremia (<1000 copies/mL) and a lower risk of maternal HIV-1 transmission (OR = 0.14, 95% CI 0.03 to 0.67, P = 0.009 and OR = 0.23, 95% CI 0.08 to 0.66, P = 0.012, respectively). CONCLUSIONS: These results demonstrate a significant relationship between genetic variants of beta-defensin-1 gene, viral load, and MTCT of HIV-1, thus supporting a critical role of innate immunity in pediatric HIV-1 infection.

Elevated human beta-defensin-2 levels indicate an activation of the innate immune system in patients with irritable bowel syndrome.
Am J Gastroenterol. 2009 Feb;104(2):404-10.
OBJECTIVES: Irritable bowel syndrome (IBS) is a highly prevalent functional disorder. According to the Rome criteria, macroscopic and histological inflammation is a crucial exclusion criterion for IBS. Human defensins appear to be part of the innate immune system in the gastrointestinal tract. Human beta-defensin-2 (HBD-2) was the first inducible human antimicrobial protein discovered. The expression is induced by probiotic microorganisms and proinflammatory cytokines. Recent results imply that HBD-2 is expressed in active intestinal inflammation, especially in ulcerative colitis (UC). Our aim was to evaluate fecal measurements of HBD-2 in patients with active UC and IBS, and in healthy controls (HCs). METHODS: Fecal specimens were collected from a total of 100 participants (30 with active UC, 46 IBS, and 24 HCs). Exclusion criteria were the current use of probiotics and antibiotics. Furthermore, IBS patients with elevated C-reactive protein or leukocytes, a history of bacterial overgrowth or infectious gastrointestinal disease over the last 6 month were excluded. Disease status was addressed in all participating subjects by medical history and current symptoms. In addition, each IBS and UC patient underwent ileocolonoscopy with histopathology. Fecal inflammation markers lactoferrin (Lf) and calprotectin (Cal) were measured by enzyme-linked immunosorbent assay (ELISA) and reported as microg/g. Fecal HBD-2 was measured by ELISA and reported as ng/g feces. In addition, immunoblots were performed for fecal HBD-2. Paraffin-embedded tissue from colonic biopsies was tested for HBD-2 peptides by immunohistochemistry. RESULTS: Lf as well as Cal was elevated in active UC (mean: 152.1+/-s.d. 374.7 microg/g; 103.5+/-87.1 microg/g), compared with IBS (8.3+/-19.4 microg/g; 18.6+/-23.3 microg/g), and HCs (0.4+/-0.5 microg/g; 7.1+/-7.9 microg/g). Scheffe post hoc tests revealed significant differences (P=0.006; P<0.001) between active UC vs. IBS and HC. In contrast, HBD-2 levels were highest in active UC (mean: 106.9+/-s.d. 91.5 ng/g), almost as high in IBS (pts 76.0+/-67.9 ng/g), and lowest for HCs (29.9+/-16.1 ng/g). Scheffe post hoc tests revealed significant differences (P<0.001) between the groups of patients (UC and IBS) vs. HCs. Immunohistochemical investigation was consistent with fecal secretion data and demonstrated the presence of beta-defensin 2 peptides in colonic epithelial enterocytes in UC as well as IBS patients with elevated fecal HBD-2. CONCLUSIONS: The results indicate significantly elevated levels of HBD-2 in patients with IBS compared with HCs and similar to those with active UC. The results support an activation of the mucosal innate defense system toward a proinflammatory response in IBS patients in the absence of macroscopic signs of inflammation.

Human Neutrophil Peptides 1-3--early markers in development of colorectal adenomas and carcinomas
Dis Markers. 2008;25(2):123-9
Expression of Human Neutrophil Peptides (HNP) 1-3 was recently found to be associated with development of colorectal cancer. Raised defensin-expression in tumours is believed to stem from increased infiltration of neutrophils into tumour environment. To further specify the role of alpha-defensins in tumourigenesis and progression, HNP1-3 were analyzed in colorectal adenomas and carcinomas of 87 patients and quantified in relation to cancer stage and grading. Using the ProteinChip arrays, HNP1-3 were found upregulated in both colorectal adenomas and carcinomas. By combining the array with Laser capture microscopy we were able to confirm that HNP1-3 are expressed by tumour cells but not by neutrophils or other tumour invading cells. These findings suggest that alpha-defensins are more likely to contribute to tumour growth than they are to mount an effective host anti-tumour response. However, the amount of HNP-expression was not found to be related to tumour stage, grading, and serological tumour markers.