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1.  An integrative pan-cancer-wide analysis of epigenetic enzymes reveals universal patterns of epigenomic deregulation in cancer 
Genome Biology  2015;16(1):140.
One of the most important recent findings in cancer genomics is the identification of novel driver mutations which often target genes that regulate genome-wide chromatin and DNA methylation marks. Little is known, however, as to whether these genes exhibit patterns of epigenomic deregulation that transcend cancer types.
Here we conduct an integrative pan-cancer-wide analysis of matched RNA-Seq and DNA methylation data across ten different cancer types. We identify seven tumor suppressor and eleven oncogenic epigenetic enzymes which display patterns of deregulation and association with genome-wide cancer DNA methylation patterns, which are largely independent of cancer type. In doing so, we provide evidence that genome-wide cancer hyper- and hypo- DNA methylation patterns are independent processes, controlled by distinct sets of epigenetic enzyme genes. Using causal network modeling, we predict a number of candidate drivers of cancer DNA hypermethylation and hypomethylation. Finally, we show that the genomic loci whose DNA methylation levels associate most strongly with expression of these putative drivers are highly consistent across cancer types.
This study demonstrates that there exist universal patterns of epigenomic deregulation that transcend cancer types, and that intra-tumor levels of genome-wide DNA hypomethylation and hypermethylation are controlled by distinct processes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0699-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4501092  PMID: 26169266
2.  Increased Mortality in Adult Trauma Patients Transfused with Blood Components Compared with Whole Blood 
Hemorrhage is a preventable cause of death among trauma patients, and management often includes transfusion, either whole blood or a combination of blood components (packed red blood cells, platelets, fresh frozen plasma). We used the 2009 National Trauma Data Bank to evaluate the relationship between transfusion type and mortality in adult major trauma patients (n = 1745). Logistic regression analysis identified three independent predictors of mortality: Injury Severity Score, emergency transfer time, and type of blood transfusion, whole blood or components. Transfusion of whole blood was associated with reduced mortality; thus, may provide superior survival outcomes in this population.
PMCID: PMC4126240  PMID: 24399315
blood transfusion; trauma; hemorrhage; mortality
3.  A BRCA1-mutation associated DNA methylation signature in blood cells predicts sporadic breast cancer incidence and survival 
Genome Medicine  2014;6(6):47.
BRCA1 mutation carriers have an 85% risk of developing breast cancer but the risk of developing non-hereditary breast cancer is difficult to assess. Our objective is to test whether a DNA methylation (DNAme) signature derived from BRCA1 mutation carriers is able to predict non-hereditary breast cancer.
In a case/control setting (72 BRCA1 mutation carriers and 72 BRCA1/2 wild type controls) blood cell DNA samples were profiled on the Illumina 27 k methylation array. Using the Elastic Net classification algorithm, a BRCA1-mutation DNAme signature was derived and tested in two cohorts: (1) The NSHD (19 breast cancers developed within 12 years after sample donation and 77 controls) and (2) the UKCTOCS trial (119 oestrogen receptor positive breast cancers developed within 5 years after sample donation and 122 controls).
We found that our blood-based BRCA1-mutation DNAme signature applied to blood cell DNA from women in the NSHD resulted in a receiver operating characteristics (ROC) area under the curve (AUC) of 0.65 (95% CI 0.51 to 0.78, P = 0.02) which did not validate in buccal cells from the same individuals. Applying the signature in blood DNA from UKCTOCS volunteers resulted in AUC of 0.57 (95% CI 0.50 to 0.64; P = 0.03) and is independent of family history or any other known risk factors. Importantly the BRCA1-mutation DNAme signature was able to predict breast cancer mortality (AUC = 0.67; 95% CI 0.51 to 0.83; P = 0.02). We also found that the 1,074 CpGs which are hypermethylated in BRCA1 mutation carriers are significantly enriched for stem cell polycomb group target genes (P <10-20).
A DNAme signature derived from BRCA1 carriers is able to predict breast cancer risk and death years in advance of diagnosis. Future studies may need to focus on DNAme profiles in epithelial cells in order to reach the AUC thresholds required of preventative measures or early detection strategies.
PMCID: PMC4110671  PMID: 25067956
4.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
PMCID: PMC3825654  PMID: 24265601
5.  Epigenetic variability in cells of normal cytology is associated with the risk of future morphological transformation 
Genome Medicine  2012;4(3):24.
Recently, it has been proposed that epigenetic variation may contribute to the risk of complex genetic diseases like cancer. We aimed to demonstrate that epigenetic changes in normal cells, collected years in advance of the first signs of morphological transformation, can predict the risk of such transformation.
We analyzed DNA methylation (DNAm) profiles of over 27,000 CpGs in cytologically normal cells of the uterine cervix from 152 women in a prospective nested case-control study. We used statistics based on differential variability to identify CpGs associated with the risk of transformation and a novel statistical algorithm called EVORA (Epigenetic Variable Outliers for Risk prediction Analysis) to make predictions.
We observed many CpGs that were differentially variable between women who developed a non-invasive cervical neoplasia within 3 years of sample collection and those that remained disease-free. These CpGs exhibited heterogeneous outlier methylation profiles and overlapped strongly with CpGs undergoing age-associated DNA methylation changes in normal tissue. Using EVORA, we demonstrate that the risk of cervical neoplasia can be predicted in blind test sets (AUC = 0.66 (0.58 to 0.75)), and that assessment of DNAm variability allows more reliable identification of risk-associated CpGs than statistics based on differences in mean methylation levels. In independent data, EVORA showed high sensitivity and specificity to detect pre-invasive neoplasia and cervical cancer (AUC = 0.93 (0.86 to 1) and AUC = 1, respectively).
We demonstrate that the risk of neoplastic transformation can be predicted from DNA methylation profiles in the morphologically normal cell of origin of an epithelial cancer. Having profiled only 0.1% of CpGs in the human genome, studies of wider coverage are likely to yield improved predictive and diagnostic models with the accuracy needed for clinical application.
Trial registration
The ARTISTIC trial is registered with the International Standard Randomised Controlled Trial Number ISRCTN25417821.
PMCID: PMC3446274  PMID: 22453031
7.  The Dynamics and Prognostic Potential of DNA Methylation Changes at Stem Cell Gene Loci in Women's Cancer 
PLoS Genetics  2012;8(2):e1002517.
Aberrant DNA methylation is an important cancer hallmark, yet the dynamics of DNA methylation changes in human carcinogenesis remain largely unexplored. Moreover, the role of DNA methylation for prediction of clinical outcome is still uncertain and confined to specific cancers. Here we perform the most comprehensive study of DNA methylation changes throughout human carcinogenesis, analysing 27,578 CpGs in each of 1,475 samples, ranging from normal cells in advance of non-invasive neoplastic transformation to non-invasive and invasive cancers and metastatic tissue. We demonstrate that hypermethylation at stem cell PolyComb Group Target genes (PCGTs) occurs in cytologically normal cells three years in advance of the first morphological neoplastic changes, while hypomethylation occurs preferentially at CpGs which are heavily Methylated in Embryonic Stem Cells (MESCs) and increases significantly with cancer invasion in both the epithelial and stromal tumour compartments. In contrast to PCGT hypermethylation, MESC hypomethylation progresses significantly from primary to metastatic cancer and defines a poor prognostic signature in four different gynaecological cancers. Finally, we associate expression of TET enzymes, which are involved in active DNA demethylation, to MESC hypomethylation in cancer. These findings have major implications for cancer and embryonic stem cell biology and establish the importance of systemic DNA hypomethylation for predicting prognosis in a wide range of different cancers.
Author Summary
DNA methylation is an important chemical modification of DNA that can affect and regulate the activity of genes in human tissue. Abnormal DNA methylation and its subsequent effects on gene activity are a hallmark of cancer, yet when precisely these DNA methylation changes occur and how they contribute to the development of cancer remains largely unexplored. In this work we measure the methylation state of DNA at over 14,000 genes in over 1,475 samples, including normal and benign cells, invasive cancers, and metastatic cancer tissue. Using cervical cancer as a model, we show that gain of abnormal methylation at genes typically un-methylated in stem cells can be detected up to 3 years in advance of the appearance of pre-cancerous cells, while those genes typically methylated in stem cells lose this methylation progressively throughout cancer development. Furthermore, we discover that this process of methylation loss during cancer progression is a marker of poor disease outcome common to all four major women-specific cancers: breast, ovarian, endometrial, and cervical cancers. Finally we demonstrate the relationship between loss of methylation and cancer-specific over-production of a specific protein known to play an active role in removing methylation from DNA. Taken together these findings highlight the complex nature of DNA methylation dynamics in cancer development as well as their potential exploitation for clinical gain.
PMCID: PMC3276553  PMID: 22346766
8.  Rosiglitazone Reduces Blood Pressure in Female Dahl Salt-sensitive Rats 
Steroids  2009;75(11):794-799.
Postmenopausal women (PMW) are at greater risk for salt-sensitive hypertension and insulin resistance than premenopausal women. Peroxisome proliferator activated receptor-gamma (PPARγ) agonists reduce blood pressure (BP) and insulin resistance in humans. As in PMW, ovariectomy (OVX) increases salt sensitivity of BP and body weight in Dahl salt sensitive (DS) rats. This study addressed whether rosiglitazone (ROSI), a PPARγ agonist, attenuates salt-sensitive hypertension in intact (INT) and OVX DS rats, and if so, whether insulin resistance, nitric oxide (NO), oxidative stress, and/or renal inflammation were contributing mediators. Telemetric BP was similar in OVX and INT on low salt diet (0.3% NaCl), but was higher in OVX than INT on high salt (8% NaCl). ROSI reduced BP in OVX and INT on both low and high salt diet, but only attenuated salt sensitivity of BP in OVX. Nitrate/nitrite excretion (NOx; index of NO) was similar in INT and OVX on low salt diet, and ROSI increased NOx in both groups. High salt diet increased NOx in all groups but ROSI only increased NOx in OVX rats. OVX females exhibited insulin resistance, increases in body weight, plasma leptin, cholesterol, numbers of renal cortical macrophages, and renal MCP-1 and osteopontin mRNA expression compared to INT. ROSI reduced cholesterol and macrophage infiltration in OVX, but not INT. In summary, PPAR-gamma activation reduces BP in INT and OVX females, but attenuates the salt sensitivity of BP in OVX only likely due to increases in NO and in part to reductions in renal resident macrophages and inflammation.
PMCID: PMC2891303  PMID: 19883672
ovariectomy; menopause; hypertension; inflammation; oxidative stress
9.  Toll-Like Receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors 
Cancer science  2010;101(4):1059-1066.
Toll-like-receptor-9 (TLR9) activates the innate immune response when exposed to non-methylated CpG-DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n=18) versus age-matched tumor free women (n=18). In breast cancer cell lines and tissues, TLR9 mRNA-expression was associated with estrogen-receptor (ER) status (n=124, P=0.005). Expression also correlated with increasing tumor grade in both breast (P=0.03) and ovarian cancer specimens (n=138, P=0.04). Immunohistochemical analysis of formalin-fixed paraffin embedded (FFPE) breast cancer tissues revealed higher TLR9 protein-expression in hormone-receptor (HR) negative specimens (n=116, P=<0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG-DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor Kappa B (NF-κB) activity with TLR9 mRNA-expression (correlation coefficient r=0.7, P<0.001). Finally, immunofluorescence analysis of BT-20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG-DNA with TLR9, which diminished when the cells were exposed to methylated CpG-DNA (mCpG-DNA) or control GpC-DNA.
In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9-overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cells lines.
PMCID: PMC3188854  PMID: 20156214
TLR9; Breast cancer; Ovarian cancer; Hormone receptors
10.  The TNF-α antagonist etanercept decreases blood pressure and protects the kidney in a mouse model of systemic lupus erythematosus 
Hypertension  2010;56(4):643-649.
Chronic inflammation has been implicated in the pathology of hypertension; however, the role for specific cytokines remains unclear. We tested whether TNF-α blockade with etanercept (Etan) reduces mean arterial pressure (MAP) in a female mouse model of systemic lupus erythematosus (SLE). SLE is a chronic inflammatory disorder with prevalent hypertension. Thirty week old SLE (NZBWF1) and control mice (NZW/LacJ) received Etan (0.8mg/kg SC weekly) for 4 weeks or vehicle. MAP (mmHg) was increased in SLE mice (150±5 vs. 113±5 in controls, p<0.05) and was lower in Etan treated SLE mice (132±3) but not controls (117±5). Albuminuria (µg/mg creatinine) was elevated in SLE mice (28742±9032 vs. 1075±883 p<0.05) and was lower in Etan treated SLE mice (8154±3899) but not control animals (783±226). Glomerulosclerosis (% of glomeruli) was evident in SLE mice (2.5±1.6 vs. 0.0±0.0 in controls, p<0.05) and was ameliorated in Etan treated SLE mice (0.1±0.1). Renal cortex CD68+ cell staining (% area) was elevated in SLE mice (4.75±0.80 vs. 0.79±0.12 in controls, p<0.05) and was lower in Etan treated SLE mice (2.28±0.32) but not controls (1.43±0.25). Renal cortex NADPH oxidase activity (RLU/mg of protein) was higher in SLE mice compared to controls (10718±1276 vs. 7584±229, p<0.05) and lowered in Etan treated SLE mice (6645±490). Renal cortex NFκB (phosphorylated and non-phosphorylated) was increased in SLE mice compared to controls and lower in Etan treated SLE mice. These data suggest that TNF-α mechanistically contributes to the development of hypertension in a chronic inflammatory disease through increased renal NFκB, oxidative stress and inflammation.
PMCID: PMC2989495  PMID: 20696988
Systemic Lupus Erythematosus; Hypertension; Inflammation; TNF-α-Oxidative Stress; cytokine
11.  A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria 
Nature  2010;468(7322):439-442.
Summary paragraph
Bacteria have developed mechanisms to communicate and compete with one another in diverse environments 1. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli 2. CDI is mediated by the CdiB/CdiA two-partner secretion system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional immunity protein (CdiI) protects CDI+ cells from autoinhibition 2, 3. The mechanisms by which CDI blocks cell growth and CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are utilized during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells in both laboratory media and upon a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network that plays an important role in bacterial competition.
PMCID: PMC3058911  PMID: 21085179
12.  An Epigenetic Signature in Peripheral Blood Predicts Active Ovarian Cancer 
PLoS ONE  2009;4(12):e8274.
Recent studies have shown that DNA methylation (DNAm) markers in peripheral blood may hold promise as diagnostic or early detection/risk markers for epithelial cancers. However, to date no study has evaluated the diagnostic and predictive potential of such markers in a large case control cohort and on a genome-wide basis.
Principal Findings
By performing genome-wide DNAm profiling of a large ovarian cancer case control cohort, we here demonstrate that active ovarian cancer has a significant impact on the DNAm pattern in peripheral blood. Specifically, by measuring the methylation levels of over 27,000 CpGs in blood cells from 148 healthy individuals and 113 age-matched pre-treatment ovarian cancer cases, we derive a DNAm signature that can predict the presence of active ovarian cancer in blind test sets with an AUC of 0.8 (95% CI (0.74–0.87)). We further validate our findings in another independent set of 122 post-treatment cases (AUC = 0.76 (0.72–0.81)). In addition, we provide evidence for a significant number of candidate risk or early detection markers for ovarian cancer. Furthermore, by comparing the pattern of methylation with gene expression data from major blood cell types, we here demonstrate that age and cancer elicit common changes in the composition of peripheral blood, with a myeloid skewing that increases with age and which is further aggravated in the presence of ovarian cancer. Finally, we show that most cancer and age associated methylation variability is found at CpGs located outside of CpG islands.
Our results underscore the potential of DNAm profiling in peripheral blood as a tool for detection or risk-prediction of epithelial cancers, and warrants further in-depth and higher CpG coverage studies to further elucidate this role.
PMCID: PMC2793425  PMID: 20019873
13.  Endotoxin, Capsule, and Bacterial Attachment Contribute to Neisseria meningitidis Resistance to the Human Antimicrobial Peptide LL-37▿ †  
Journal of Bacteriology  2009;191(12):3861-3868.
Pathogenic bacteria have evolved numerous mechanisms to evade the human immune system and have developed widespread resistance to traditional antibiotics. We studied the human pathogen Neisseria meningitidis and present evidence of novel mechanisms of resistance to the human antimicrobial peptide LL-37. We found that bacteria attached to host epithelial cells are resistant to 10 μM LL-37 whereas bacteria in solution or attached to plastic are killed, indicating that the cell microenvironment protects bacteria. The bacterial endotoxin lipooligosaccharide and the polysaccharide capsule contribute to LL-37 resistance, probably by preventing LL-37 from reaching the bacterial membrane, as more LL-37 reaches the bacterial membrane on both lipooligosaccharide-deficient and capsule-deficient mutants whereas both mutants are also more susceptible to LL-37 killing than the wild-type strain. N. meningitidis bacteria respond to sublethal doses of LL-37 and upregulate two of their capsule genes, siaC and siaD, which further results in upregulation of capsule biosynthesis.
PMCID: PMC2698406  PMID: 19376861
14.  The ScpC Protease of Streptococcus pyogenes Affects the Outcome of Sepsis in a Murine Model ▿  
Infection and Immunity  2008;76(9):3959-3966.
The ScpC protease of Streptococcus pyogenes degrades interleukin-8 (IL-8), a chemokine that mediates neutrophil transmigration and activation. The ability to degrade IL-8 differs dramatically among clinical isolates of S. pyogenes. Bacteria expressing ScpC overcome immune clearance by preventing the recruitment of neutrophils in soft tissue infection of mice. To study the role of ScpC in streptococcal sepsis, we generated an ScpC mutant that did not degrade IL-8 and thus failed to prevent the recruitment of immune cells as well as to cause disease after soft tissue infection. In a murine model of sepsis, challenge with the ScpC mutant resulted in more severe systemic disease with higher bacteremia levels and mortality than did challenge with the wild-type strain. As expected, the blood level of KC, the murine IL-8 homologue, increased in mice infected with the ScpC mutant. However, the elevated KC levels did not influence neutrophil numbers in blood, as it did in soft tissue, indicating that additional factors contributed to neutrophil transmigration in blood. In addition, the absence of ScpC increased tumor necrosis factor, IL-6, and C5a levels in blood, which contributed to disease severity. Thus, the ScpC mutant triggers high neutrophil infiltration but not lethal outcome after soft tissue infection, whereas intravenous infection leads to highly aggressive systemic disease.
PMCID: PMC2519448  PMID: 18573900
15.  Induction of the Antimicrobial Peptide CRAMP in the Blood-Brain Barrier and Meninges after Meningococcal Infection▿  
Infection and Immunity  2006;74(12):6982-6991.
Antimicrobial peptides are present in most living species and constitute important effector molecules of innate immunity. Recently, we and others have detected antimicrobial peptides in the brain. This is an organ that is rarely infected, which has mainly been ascribed to the protective functions of the blood-brain barrier (BBB) and meninges. Since the bactericidal properties of the BBB and meninges are not known, we hypothesized that antimicrobial peptides could play a role in these barriers. We addressed this hypothesis by infecting mice with the neuropathogenic bacterium Neisseria meningitidis. Brains were analyzed for expression of the antimicrobial peptide CRAMP by immunohistochemistry in combination with confocal microscopy. After infection, we observed induction of CRAMP in endothelial cells of the BBB and in cells of the meninges. To explore the functional role of CRAMP in meningococcal disease, we infected mice deficient of the CRAMP gene. Even though CRAMP did not appear to protect the brain from invasion of meningococci, CRAMP knockout mice were more susceptible to meningococcal infection than wild-type mice and exhibited increased meningococcal growth in blood, liver, and spleen. Moreover, we could demonstrate that carbonate, a compound that accumulates in the circulation during metabolic acidosis, makes meningococci more susceptible to CRAMP.
PMCID: PMC1698100  PMID: 17030578

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