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1.  Characterization of the Mouse Beta Defensin 1, Defb1, Mutant Mouse Model  
Infection and Immunity  2002;70(6):3053-3060.
Beta defensins are small cationic antimicrobial peptides present in the respiratory system which have been proposed to be dysfunctional in the environment of the cystic fibrosis lung. Defb1, a murine homologue to the human beta defensins, has also been found to be expressed in the respiratory system and, in order to examine the function of beta defensins in vivo, gene targeting was used to generate Defb1-deficient (Defb1tm1Hgu/Defb1tm1Hgu [Defb1−/−]) mice. The Defb1 synthetic peptide was shown to have a salt-sensitive antimicrobial activity that was stronger against Staphylococcus aureus than against Escherichia coli or Pseudomonas aeruginosa. Defb1−/− mice were found, however, to be effective in the clearance of the cystic fibrosis relevant pathogen S. aureus from the airways after nebulization. Although no overt deleterious phenotype was evident in the Defb1−/− mice, the number of mutant mice found to harbor bacteria of the Staphylococcus species in the bladder was significantly higher (P = 0.008) than that of controls, suggesting a role for these peptides in resistance to urinary tract infection.
PMCID: PMC128030  PMID: 12010997
2.  Increased Expression of Beta-Defensin 1 (DEFB1) in Chronic Obstructive Pulmonary Disease 
PLoS ONE  2011;6(7):e21898.
On-going airway inflammation is characteristic for the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the key factors determining the decrease in lung function, an important clinical parameter of COPD, are not clear. Genome-wide linkage analyses provide evidence for significant linkage to airway obstruction susceptibility loci on chromosome 8p23, the location of the human defensin gene cluster. Moreover, a genetic variation in the defensin beta 1 (DEFB1) gene was found to be associated with COPD. Therefore, we hypothesized that DEFB1 is differently regulated and expressed in human lungs during COPD progression. Gene expression of DEFB1 was assessed in bronchial epithelium and BAL fluid cells of healthy controls and patients with COPD and using bisulfite sequencing and ChIP analysis, the epigenetic control of DEFB1 mRNA expression was investigated. We can demonstrate that DEFB1 mRNA expression was significantly increased in bronchopulmonary specimen of patients with COPD (n = 34) vs. healthy controls (n = 10) (p<0.0001). Furthermore, a significant correlation could be detected between DEFB1 and functional parameters such as FEV1 (p = 0.0024) and the FEV1/VC ratio (p = 0.0005). Upregulation of DEFB1 mRNA was paralleled by changes in HDAC1-3, HDAC5 and HDAC8 mRNA expression. Whereas bisulfite sequencing revealed no differences in the methylation state of DEFB1 promoter between patients with COPD and controls, ChIP analysis showed that enhanced DEFB1 mRNA expression was associated with the establishment of an active histone code. Thus, expression of human DEFB1 is upregulated and related to the decrease in pulmonary function in patients with COPD.
PMCID: PMC3139569  PMID: 21818276
3.  8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations 
BMC Genomics  2014;15:64.
The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination.
We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes.
MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.
PMCID: PMC3937106  PMID: 24460793
Beta-defensin; Copy number variation; Quantitative real-time PCR; Paralog ratio tests; Multiplex ligation-dependent probe amplification; Clustering; Pseudogene; Low frequency sequence variations
4.  Copy Number Variation of the Beta-Defensin Genes in Europeans: No Supporting Evidence for Association with Lung Function, Chronic Obstructive Pulmonary Disease or Asthma 
PLoS ONE  2014;9(1):e84192.
Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02–1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72–1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.
PMCID: PMC3880289  PMID: 24404154
5.  Histone Deacetylase (HDAC) 1 Controls the Expression of Beta Defensin 1 in Human Lung Epithelial Cells 
PLoS ONE  2012;7(11):e50000.
Deregulation of the expression human beta defensin 1 (DEFB1), an antimicrobial peptide, has been implicated in the pathogenesis of COPD and asthma. Since the molecular mechanisms that regulate DEFB1 gene expression are widely unknown, the epigenetic processes involved in the regulation of the constitutive expression of DEFB1 in lung epithelial cells (A549) were investigated. The data demonstrate that histone deacetylases (HDACs) participate in the regulation of DEFB1 gene expression. Inhibition of the class I HDACs, HDACs 1-3, increases DEFB1 gene expression in A549 cells. Chromatin immunoprecipitation (ChIP) assays revealed that the inhibition of the class I HDACs also results in modifications of the chromatin at the DEFB1 promoter. Histone modifications, histone H3 acetylation and H3K4 trimethylation, that are associated with transcriptional activation, were found to increase after inhibition of HDACs 1-3. Finally, RNAi knockdown experiments identified HDAC1 as the sole HDAC responsible for maintaining the constitutive level of DEFB1 transcription. Taken together, our data reveal epigenetic mechanisms which are the basis of the maintenance of the constitutive gene expression of human beta defensin 1.
PMCID: PMC3502185  PMID: 23185513
6.  The signature of long-standing balancing selection at the human defensin β-1 promoter 
Genome Biology  2008;9(9):R143.
Analysis of the human beta defensin 1 promoter region in six human populations reveals a signature of balancing selection.
Defensins, small endogenous peptides with antimicrobial activity, are pivotal components of the innate immune response. A large cluster of defensin genes is located on human chromosome 8p; among them the beta defensin 1 (DEFB1) promoterhas been extensively studied since discovery that specific polymorphisms and haplotypes associate with asthma and atopy, susceptibility to severe sepsis, as well as HIV and Candida infection predisposition.
Here, we characterize the sequence variation and haplotype structure of the DEFB1 promoter region in six human populations. In all of them, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, reduced population differentiation and a genealogy with common haplotypes separated by deep branches. Indeed, a significant departure from the expectation of evolutionary neutrality was observed in all populations and the possibility that this is due to demographic history alone was ruled out. Also, we verified that the selection signature is restricted to the promoter region and not due to a linked balanced polymorphism. A phylogeny-based estimation indicated that the two major haplotype clades separated around 4.5 million years ago, approximately the time when the human and chimpanzee lineages split.
Altogether, these features represent strong molecular signatures of long-term balancing selection, a process that is thought to be extremely rare outside major histocompatibility complex genes. Our data indicate that the DEFB1 promoter region carries functional variants and support previous hypotheses whereby alleles predisposing to atopic disorders are widespread in modern societies because they conferred resistance to pathogens in ancient settings.
PMCID: PMC2592704  PMID: 18817538
7.  Haplotyping and copy number estimation of the highly polymorphic human beta-defensin locus on 8p23 by 454 amplicon sequencing 
BMC Genomics  2010;11:252.
The beta-defensin gene cluster (DEFB) at chromosome 8p23.1 is one of the most copy number (CN) variable regions of the human genome. Whereas individual DEFB CNs have been suggested as independent genetic risk factors for several diseases (e.g. psoriasis and Crohn's disease), the role of multisite sequence variations (MSV) is less well understood and to date has only been reported for prostate cancer. Simultaneous assessment of MSVs and CNs can be achieved by PCR, cloning and Sanger sequencing, however, these methods are labour and cost intensive as well as prone to methodological bias introduced by bacterial cloning. Here, we demonstrate that amplicon sequencing of pooled individual PCR products by the 454 technology allows in-depth determination of MSV haplotypes and estimation of DEFB CNs in parallel.
Six PCR products spread over ~87 kb of DEFB and harbouring 24 known MSVs were amplified from 11 DNA samples, pooled and sequenced on a Roche 454 GS FLX sequencer. From ~142,000 reads, ~120,000 haplotype calls (HC) were inferred that identified 22 haplotypes ranging from 2 to 7 per amplicon. In addition to the 24 known MSVs, two additional sequence variations were detected. Minimal CNs were estimated from the ratio of HCs and compared to absolute CNs determined by alternative methods. Concordance in CNs was found for 7 samples, the CNs differed by one in 2 samples and the estimated minimal CN was half of the absolute in one sample. For 7 samples and 2 amplicons, the 454 haplotyping results were compared to those by cloning/Sanger sequencing. Intrinsic problems related to chimera formation during PCR and differences between haplotyping by 454 and cloning/Sanger sequencing are discussed.
Deep amplicon sequencing using the 454 technology yield thousands of HCs per amplicon for an affordable price and may represent an effective method for parallel haplotyping and CN estimation in small to medium-sized cohorts. The obtained haplotypes represent a valuable resource to facilitate further studies of the biomedical impact of highly CN variable loci such as the beta-defensin locus.
PMCID: PMC2873476  PMID: 20403190
8.  Association studies of the copy-number variable ß-defensin cluster on 8p23.1 in adenocarcinoma and chronic pancreatitis 
BMC Research Notes  2012;5:629.
Human ß-defensins are a family of antimicrobial peptides located at the mucosal surface. Both sequence multi-site variations (MSV) and copy-number variants (CNV) of the defensin-encoding genes are associated with increased risk for various diseases, including cancer and inflammatory conditions such as psoriasis and acute pancreatitis. In a case–control study, we investigated the association between MSV in DEFB104 as well as defensin gene (DEF) cluster copy number (CN), and pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP).
Two groups of PDAC (N=70) and CP (N=60) patients were compared to matched healthy control groups CARLA1 (N=232) and CARLA2 (N=160), respectively. Four DEFB104 MSV were haplotyped by PCR, cloning and sequencing. DEF cluster CN was determined by multiplex ligation-dependent probe amplification.
Neither the PDAC nor the CP cohorts show significant differences in the DEFB104 haplotype distribution compared to the respective control groups CARLA1 and CARLA2, respectively.
The diploid DEF cluster CN exhibit a significantly different distribution between PDAC and CARLA1 (Fisher’s exact test P=0.027), but not between CP and CARLA2 (P=0.867).
Different DEF cluster b CN distribution between PDAC patients and healthy controls indicate a potential protective effect of higher CNs against the disease.
PMCID: PMC3532138  PMID: 23148552
Defensins; Single nucleotide variants; Copy number variation; Chronic pancreatitis; Pancreatic ductal adenocarcinoma
9.  Narrowing down the distal border of the copy number variable beta-defensin gene cluster on human 8p23 
BMC Research Notes  2014;7:93.
Copy number variation (CNV) in the range from 2 to 12 per diploid genome is an outstanding feature of the beta-defensin gene (DEFB) cluster on human chromosome 8p23.1 numerously demonstrated by different methods. So far, CNV was proven for a 115 kb region between DEFB4 and 21 kb proximal of DEFB107 but the borders for the entire CNV repeat unit are still unknown. Our study aimed to narrow down the distal border of the DEFB cluster.
We established tests for length polymorphisms based on amplification and capillary electrophoresis with laser-induced fluorescence (CE-LIF) analysis of seven insertion/deletion (indel) containing regions spread over the entire cluster. The tests were carried out with 25 genomic DNAs with different previously determined cluster copy numbers. CNV was demonstrated for six indels between ~1 kb distal of DEFB108P and 10 kb proximal of DEFB107. In contrast, the most distal indel is not affected by CNV.
Our analysis fixes the minimal length of proven CNV to 157 kb including DEFB108P but excluding DEFB109P. The distal border between CNV and non-CNV part of the DEF cluster is located in the 59 kb interval chr8:7,171,082-7,230,128.
PMCID: PMC3942070  PMID: 24552181
Defensin; CNV borders; Length polymorphism; Paralog ratio test; Indel; DEFB108P; DEFB109P
10.  Directional and balancing selection in human beta-defensins 
In primates, infection is an important force driving gene evolution, and this is reflected in the importance of infectious disease in human morbidity today. The beta-defensins are key components of the innate immune system, with antimicrobial and cell signalling roles, but also reproductive functions. Here we examine evolution of beta-defensins in catarrhine primates and variation within different human populations.
We show that five beta-defensin genes that do not show copy number variation in humans show evidence of positive selection in catarrhine primates, and identify specific codons that have been under selective pressure. Direct haplotyping of DEFB127 in humans suggests long-term balancing selection: there are two highly diverged haplotype clades carrying different variants of a codon that, in primates, is positively selected. For DEFB132, we show that extensive diversity, including a four-state amino acid polymorphism (valine, isoleucine, alanine and threonine at position 93), is present in hunter-gatherer populations, both African and non-African, but not found in samples from agricultural populations.
Some, but not all, beta-defensin genes show positive selection in catarrhine primates. There is suggestive evidence of different selective pressures on these genes in humans, but the nature of the selective pressure remains unclear and is likely to differ between populations.
PMCID: PMC2373304  PMID: 18416833
11.  Association of Mannose-Binding Lectin 2 Gene Polymorphisms with Persistent Staphylococcus aureus Bacteremia 
PLoS ONE  2014;9(3):e89139.
Mannose-binding lectin (MBL) is an important component of innate immunity. Structural and promoter polymorphisms in the MBL2 gene that are responsible for low MBL levels are associated with susceptibility to infectious diseases. The objective of this study was to investigate the association of serum MBL levels and MBL2 polymorphisms with persistent Staphylococcus aureus bacteremia (SAB) in adult Korean patients.
We conducted a case-control study nested in a prospective cohort of patients with SAB. The study compared 41 patients with persistent bacteremia (≥7 days) and 46 patients with resolving bacteremia (<3 days). In each subject, we genotyped six single-nucleotide polymorphisms in the promoter region (alleles H/L, X/Y, and P/Q) and exon 1 (alleles A/B, A/C, and A/D) of the MBL2 gene and measured serum MBL concentrations. We also compared MBL2 genotypes between SAB patients and healthy people.
Patients with persistent bacteremia were significantly more likely to have low/deficient MBL-producing genotypes and resultant low serum MBL levels, than were patients with resolving bacteremia (P = 0.019 and P = 0.012, respectively). Independent risk factors for persistent bacteremia were metastatic infection (adjusted odds ratio [aOR], 34.7; 95% confidence interval [CI], 12.83–196.37; P = 0.003), methicillin resistance (aOR, 4.10; 95% CI, 3.19–29.57; P = 0.025), and low/deficient MBL-producing genotypes (aOR, 7.64; 95% CI, 4.12–63.39; P = 0.003). Such genotypes were significantly more common in patients with persistent bacteremia than in healthy people (OR, 2.09; 95% CI, 1.03–4.26; P = 0.040).
This is the first demonstration of an association of low MBL levels and MBL2 polymorphisms responsible for low or deficient MBL levels with persistent SAB. A combination of factors, including clinical and microbiological characteristics and host defense factors such as MBL levels, may together contribute to the development of persistent SAB.
PMCID: PMC3942407  PMID: 24595015
12.  Copy Number Variation within Human β-Defensin Gene Cluster Influences Progression to AIDS in the Multicenter AIDS Cohort Study 
Study background
DEFB4/103A encoding β-defensin 2 and 3, respectively, inhibit CXCR4-tropic (X4) viruses in vitro. We determined whether DEFB4/103A Copy Number Variation (CNV) influences time-to-X4 and time-to-AIDS outcomes.
We utilized samples from a previously published Multicenter AIDS Cohort Study (MACS), which provides longitudinal account of viral tropism in relation to the full spectrum of rates of disease progression. Using traditional models for time-to-event analysis, we investigated association between DEFB4/103A CNV and the two outcomes, and interaction between DEFB4/103A CNV and disease progression groups, Fast and Slow.
Time-to-X4 and time-to-AIDS were weakly correlated. There was a stronger relationship between these two outcomes for the fast progressors. DEFB4/103A CNV was associated with time-to-AIDS, but not time-to-X4. The association between higher DEFB4/103A CNV and time-to-AIDS was more pronounced for the slow progressors.
DEFB4/103A CNV was associated with time-to-AIDS in a disease progression group-specific manner in the MACS cohort. Our findings may contribute to enhancing current understanding of how genetic predisposition influences AIDS progression.
PMCID: PMC3610425  PMID: 23543857
AIDS progression; β-Defensin; DEFB4; DEFB103A; MACS; X4 emergence
13.  A genome-wide association study of variants associated with acquisition of Staphylococcus aureus bacteremia in a healthcare setting 
Humans vary in their susceptibility to acquiring Staphylococcus aureus infection, and research suggests that there is a genetic basis for this variability. Several recent genome-wide association studies (GWAS) have identified variants that may affect susceptibility to infectious diseases, demonstrating the potential value of GWAS in this arena.
We conducted a GWAS to identify common variants associated with acquisition of S. aureus bacteremia (SAB) resulting from healthcare contact. We performed a logistic regression analysis to compare patients with healthcare contact who developed SAB (361 cases) to patients with healthcare contact in the same hospital who did not develop SAB (699 controls), testing 542,410 SNPs and adjusting for age (by decade), sex, and 6 significant principal components from our EIGENSTRAT analysis. Additionally, we evaluated the joint effect of the host and pathogen genomes in association with severity of SAB infection via logistic regression, including an interaction of host SNP with bacterial genotype, and adjusting for age (by decade), sex, the 6 significant principal components, and dialysis status. Bonferroni corrections were applied in both analyses to control for multiple comparisons.
Ours is the first study that has attempted to evaluate the entire human genome for variants potentially involved in the acquisition or severity of SAB. Although this study identified no common variant of large effect size to have genome-wide significance for association with either the risk of acquiring SAB or severity of SAB, the variant (rs2043436) most significantly associated with severity of infection is located in a biologically plausible candidate gene (CDON, a member of the immunoglobulin family) and may warrant further study.
The genetic architecture underlying SAB is likely to be complex. Future investigations using larger samples, narrowed phenotypes, and advances in both genotyping and analytical methodologies will be important tools for identifying causative variants for this common and serious cause of healthcare-associated infection.
PMCID: PMC3928605  PMID: 24524581
Genomics; Genome-wide association study; Case–control study; Staphylococcus aureus; Bacteremia; Gram-positive bacterial infections; Polymorphism, single-nucleotide; Infections; Nosocomial; Cross infection
HIV medicine  2008;9(10):858-862.
To define the incidence and risk factors for methicillin resistant Staphylococcus aureus (MRSA) bacteremia in an urban HIV-infected population.
A retrospective cohort study and nested, case-control analyses set in an urban HIV outpatient clinic in Baltimore.
Over a four-year period (2000–2004) the incidence of Staphylococcus aureus bacteremia (SAB) was determined from an electronic database of blood culture results. Risk factors for MRSA bacteremia were assessed over a five-year period (2000–2005) using methicillin sensitive Staphylococcus aureus (MSSA) bacteremia and bacteremia-free controls.
Of 4,607 patients followed for a total of 11,020 person-years (PY) of follow-up, 216 episodes of SAB occurred (incidence: 19.6 cases per 1000 PY.) Of these, 94 cases (43.5%) were methicillin-resistant (MRSA bacteremia incidence: 8.5 per 1000 PY.) The incidence of MRSA bacteremia increased from 5.3 per 1000 PY in 2000–01 to 11.9 per 1000 PY in 2003–04 (p = 0.001). Significant risk factors for MRSA bacteremia included injection drug use (IDU) [Adjusted Odds Ratio (AOR) = 4.61 (95% CI: 2.32–20.72)], end-stage renal disease (ESRD) [7.78 (2.92–20.72)], and CD4 count <200 cells/mm3 at the event. Patients with MRSA were more likely to have ESRD [AOR = 2.89 (1.12–7.49)] and greater immunosuppression than those with MSSA bacteremia.
The incidence of MRSA bacteremia increased from 2000 to 2004 in our HIV-infected cohort. Our data suggest that initial therapy for S. aureus bacteremia in HIV-infected patients, particularly in those with MRSA bacteremia risk factors, may require antimicrobial agents active against MRSA.
PMCID: PMC2581476  PMID: 18754806
HIV; MRSA; Staphylococcus aureus; Bacteremia
15.  Antimicrobial Activity of Murine Lung Cells against Staphylococcus aureus Is Increased In Vitro and In Vivo after Elafin Gene Transfer  
Infection and Immunity  2005;73(6):3609-3617.
Staphylococcus aureus is a pathogen often found in pneumonia and sepsis. In the context of the resistance of this organism to conventional antibiotics, an understanding of the regulation of natural endogenous antimicrobial molecules is of paramount importance. Previous studies have shown that both human and mouse airways express a variety of these molecules, including defensins, cathelicidins, and the four-disulfide core protein secretory leukocyte protease inhibitor. We demonstrate here by culturing mouse tracheal epithelial cells at an air-liquid interface that, despite the production of Defb1, Defb14, and Defr1 in this system, these cells are unable to clear S. aureus when exposed to this respiratory pathogen. Using an adenovirus (Ad)-mediated gene transfer strategy, we show that overexpression of elafin, an anti-elastase/antimicrobial molecule (also a member of the four-disulfide core protein family), dramatically improves the clearance of S. aureus. In addition, we also demonstrate that this overexpression is efficient in vivo and that intratracheal instillation of Ad-elafin significantly reduced the lung bacterial load and demonstrates concomitant anti-inflammatory activity by reducing neutrophil numbers and markers of lung inflammation, such as bronchoalveolar lavage levels of tumor necrosis factor and myeloperoxidase. These findings show that an increased antimicrobial activity phenotype is provided by the elafin molecule and have implications for its use in S. aureus-associated local and systemic infections.
PMCID: PMC1111862  PMID: 15908390
16.  Stable incidence and continued improvement in short term mortality of Staphylococcus aureus bacteraemia between 1995 and 2008 
BMC Infectious Diseases  2012;12:260.
The objective of this study was to assess temporal changes in incidence and short term mortality of Staphylococcus aureus bacteraemia (SAB) from 1995 through 2008.
The study was conducted as a nation-wide observational cohort study with matched population controls. The setting was hospitalized patients in Denmark 1995-2008. Uni- and multivariate analyses were used to analyze the hazard of death within 30 days from SAB.
A total of 16 330 cases of SAB were identified: 57% were hospital-associated (HA), 31% were community-acquired (CA) and 13% were of undetermined acquisition. The overall adjusted incidence rate remained stable at 23 per 100 000 population but the proportion of SAB cases older than 75 years increased significantly. Comorbidity in the cohort as measured by Charlson comorbidity index (CCI) score and alcohol-related diagnoses increased over the study period. In contrast, among the population controls the CCI remained stable and alcohol-related diagnoses increased slightly. For HA SAB crude 30-day mortality decreased from 27.8% to 21.8% (22% reduction) whereas the change for CA SAB was small (26.5% to 25.8%). By multivariate Cox regression, age, female sex, time period, CCI score and alcohol-related diagnoses were associated with increased mortality regardless of mode of acquisition.
Throughout a 14-year period the overall incidence of SAB remained stable while the overall short term prognosis continued to improve despite increased age and accumulation of comorbidity in the cohort. However, age and comorbidity were strong prognostic indicators for short term mortality.
PMCID: PMC3507819  PMID: 23075215
Bacteraemia; Epidemiology; Incidence; Mortality; Comorbidity; Alcoholism; Staphylococcus aureus; Charlson comorbidity index
17.  Beta-defensin genomic copy number is not a modifier locus for cystic fibrosis 
Human beta-defensin 2 (DEFB4, also known as DEFB2 or hBD-2) is a salt-sensitive antimicrobial protein that is expressed in lung epithelia. Previous work has shown that it is encoded in a cluster of beta-defensin genes at 8p23.1, which varies in copy number between 2 and 12 in different individuals. We determined the copy number of this locus in 355 patients with cystic fibrosis (CF), and tested for correlation between beta-defensin cluster genomic copy number and lung disease associated with CF. No significant association was found.
PMCID: PMC1318481  PMID: 16336654
18.  A Worldwide Analysis of Beta-Defensin Copy Number Variation Suggests Recent Selection of a High-Expressing DEFB103 Gene Copy in East Asia 
Human Mutation  2011;32(7):743-750.
Beta-defensins are a family of multifunctional genes with roles in defense against pathogens, reproduction, and pigmentation. In humans, six beta-defensin genes are clustered in a repeated region which is copy-number variable (CNV) as a block, with a diploid copy number between 1 and 12. The role in host defense makes the evolutionary history of this CNV particularly interesting, because morbidity due to infectious disease is likely to have been an important selective force in human evolution, and to have varied between geographical locations. Here, we show CNV of the beta-defensin region in chimpanzees, and identify a beta-defensin block in the human lineage that contains rapidly evolving noncoding regulatory sequences. We also show that variation at one of these rapidly evolving sequences affects expression levels and cytokine responsiveness of DEFB103, a key inhibitor of influenza virus fusion at the cell surface. A worldwide analysis of beta-defensin CNV in 67 populations shows an unusually high frequency of high-DEFB103-expressing copies in East Asia, the geographical origin of historical and modern influenza epidemics, possibly as a result of selection for increased resistance to influenza in this region. Hum Mutat 32:743–750, 2011. © 2011 Wiley-Liss, Inc.
PMCID: PMC3263423  PMID: 21387465
CNV; defensin; antimicrobial; influenza; paralogue ratio test
19.  DUX4 activates germline genes, retroelements and immune-mediators: Implications for facioscapulohumeral dystrophy 
Developmental Cell  2011;22(1):38-51.
Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.
PMCID: PMC3264808  PMID: 22209328
20.  β-Defensin Genomic Copy Number Does Not Influence the Age of Onset in Huntington’s Disease 
Journal of Huntington's disease  2013;2(1):107-124.
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by the abnormal expansion of a CAG triplet repeat tract in the huntingtin gene. While the length of this CAG expansion is the major determinant of the age of onset (AO), other genetic factors have also been shown to play a modulatory role. Recent evidence suggests that neuroinflammation is a pivotal factor in the pathogenesis of HD, and that targeting this process may have important therapeutic ramifications. The human β-defensin 2 (hBD2) – encoded by DEFB4 – is an antimicrobial peptide that exhibits inducible expression in astrocytes during inflammation and is an important regulator of innate and adaptive immune response. Therefore, DEFB4 may contribute to the neuroinflammatory processes observed in HD.
In this study we tested the hypothesis that copy number variation (CNV) of the β-defensin region, including DEFB4, modifies the AO in HD.
Methods and results
We genotyped β-defensin CNV in 490 HD individuals using the paralogue ratio test and found no association between β-defensin CNV and onset of HD.
We conclude that it is unlikely that DEFB4 plays a role in HD pathogenesis.
PMCID: PMC3935171  PMID: 24587836
Genetic modifier; copy number variation; inflammation
21.  Staphylococcus aureus Bacteremia, Australia 
Emerging Infectious Diseases  2005;11(4):554-561.
S. aureus bacteremia in Australia is increasingly caused by MRSA, which is likely to affect empiric prescribing of antimicrobial drugs in suspected cases.
Staphylococcus aureus bacteremia (SAB) is common and increasing worldwide. A retrospective review was undertaken to quantify the number of cases, their place of acquisition, and the proportions caused by methicillin-resistant S. aureus (MRSA) in 17 hospitals in Australia. Of 3,192 episodes, 1,571 (49%) were community onset. MRSA caused 40% of hospital-onset episodes and 12% of community-onset episodes. The median rate of SAB was 1.48/1,000 admissions (range 0.61–3.24; median rate for hospital-onset SAB was 0.7/1,000 and for community onset 0.8/1,000 admissions). Using these rates, we estimate that ≈6,900 episodes of SAB occur annually in Australia (35/100,000 population). SAB is common, and a substantial proportion of cases may be preventable. The epidemiology is evolving, with >10% of community-onset SAB now caused by MRSA. This is an emerging infectious disease concern and is likely to impact on empiric antimicrobial drug prescribing in suspected cases of SAB.
PMCID: PMC3320328  PMID: 15829193
Staphylococcus aureus; bacteremia; hospital infections; methicillin resistance; mortality; fatal outcome; nosocomial infections; infection control; indwelling catheters
22.  Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses 
PLoS ONE  2009;4(6):e5810.
Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.
PMCID: PMC2686169  PMID: 19503839
23.  Single-Nucleotide Polymorphisms (SNPs) in Human β-Defensin 1: High-Throughput SNP Assays and Association with Candida Carriage in Type I Diabetics and Nondiabetic Controls 
β-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human β-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as ≤350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5′ untranslated region (C→G at position −44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C→G SNP at position −44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.
PMCID: PMC149626  PMID: 12517831
24.  Predictors of Mortality in Staphylococcus aureus Bacteremia 
Clinical Microbiology Reviews  2012;25(2):362-386.
Summary: Staphylococcus aureus bacteremia (SAB) is an important infection with an incidence rate ranging from 20 to 50 cases/100,000 population per year. Between 10% and 30% of these patients will die from SAB. Comparatively, this accounts for a greater number of deaths than for AIDS, tuberculosis, and viral hepatitis combined. Multiple factors influence outcomes for SAB patients. The most consistent predictor of mortality is age, with older patients being twice as likely to die. Except for the presence of comorbidities, the impacts of other host factors, including gender, ethnicity, socioeconomic status, and immune status, are unclear. Pathogen-host interactions, especially the presence of shock and the source of SAB, are strong predictors of outcomes. Although antibiotic resistance may be associated with increased mortality, questions remain as to whether this reflects pathogen-specific factors or poorer responses to antibiotic therapy, namely, vancomycin. Optimal management relies on starting appropriate antibiotics in a timely fashion, resulting in improved outcomes for certain patient subgroups. The roles of surgery and infectious disease consultations require further study. Although the rate of mortality from SAB is declining, it remains high. Future international collaborative studies are required to tease out the relative contributions of various factors to mortality, which would enable the optimization of SAB management and patient outcomes.
PMCID: PMC3346297  PMID: 22491776
25.  Effect of Statin Therapy in the Outcome of Bloodstream Infections Due to Staphylococcus aureus: A Prospective Cohort Study 
PLoS ONE  2013;8(12):e82958.
Statins have pleiotropic effects that could influence the prevention and outcome of some infectious diseases. There is no information about their specific effect on Staphylococcus aureus bacteremia (SAB).
A prospective cohort study including all SAB diagnosed in patients aged ≥18 years admitted to a 950-bed tertiary hospital from March 2008 to January 2011 was performed. The main outcome variable was 14-day mortality, and the secondary outcome variables were 30-day mortality, persistent bacteremia (PB) and presence of severe sepsis or septic shock at diagnosis of SAB. The effect of statin therapy at the onset of SAB was studied by multivariate logistic regression and Cox regression analysis, including a propensity score for statin therapy.
We included 160 episodes. Thirty-three patients (21.3%) were receiving statins at the onset of SAB. 14-day mortality was 21.3%. After adjustment for age, Charlson index, Pitt score, adequate management, and high risk source, statin therapy had a protective effect on 14-day mortality (adjusted OR = 0.08; 95% CI: 0.01–0.66; p = 0.02), and PB (OR = 0.89; 95% CI: 0.27–1.00; p = 0.05) although the effect was not significant on 30-day mortality (OR = 0.35; 95% CI: 0.10–1.23; p = 0.10) or presentation with severe sepsis or septic shock (adjusted OR = 0.89; CI 95%: 0.27–2.94; p = 0.8). An effect on 30-day mortality could neither be demonstrated on Cox analysis (adjusted HR = 0.5; 95% CI: 0.19–1.29; p = 0.15).
Statin treatment in patients with SAB was associated with lower early mortality and PB. Randomized studies are necessary to identify the role of statins in the treatment of patients with SAB.
PMCID: PMC3871563  PMID: 24376617

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