Postdiagnosis circulating osteoprotegerin and TRAIL concentrations and survival and recurrence after a breast cancer diagnosis: results from the MARIE patient cohort.

BACKGROUND: Experimental studies suggest a role for osteoprotegerin (OPG) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in mammary tumor development and progression. These biomarkers have been minimally investigated with respect to outcomes in breast cancer patients. METHODS: OPG and TRAIL were evaluated in blood samples collected from 2459 breast cancer patients enrolled in the MARIE study, a prospective population-based patient cohort, at median of 129 days after diagnosis. Participants were between ages 50 and 74 at diagnosis and recruited from 2002 to 2005 in two regions of Germany. Follow-up for recurrence and mortality was conducted through June 2015. Delayed-entry Cox proportional hazards regression was used to assess associations between OPG and TRAIL with all-cause and breast cancer-specific mortality, and recurrence, both overall and by tumor hormone receptor status. RESULTS: Median follow-up time was 11.7 years, with 485 deaths reported (277 breast cancer-specific). Higher OPG concentrations were associated with a higher risk of all-cause mortality (hazard ratio for 1-unit log2-transformed concentration (HRlog2) = 1.24 (95% confidence interval 1.03-1.49). Associations were observed in women diagnosed with ER-PR- tumors or discordant hormone receptor status (ER-PR-, HRlog2 = 1.93 (1.20-3.10); discordant ERPR, 1.70 (1.03-2.81)), but not for women with ER + PR + tumors (HRlog2 = 1.06 (0.83-1.35)). OPG was associated with a higher risk of recurrence among women with ER-PR- disease (HRlog2 = 2.18 (1.39-3.40)). We observed no associations between OPG and breast cancer-specific survival, or for TRAIL and any outcome. CONCLUSIONS: Higher circulating OPG may be a biomarker of a higher risk of poor outcome among women diagnosed with ER- breast cancer. Further mechanistic studies are warranted.

Calorie restriction improves metabolic state independently of gut microbiome composition: a randomized dietary intervention trial.

BACKGROUND: The gut microbiota has been suggested to play a significant role in the development of overweight and obesity. However, the effects of calorie restriction on gut microbiota of overweight and obese adults, especially over longer durations, are largely unexplored. METHODS: Here, we longitudinally analyzed the effects of intermittent calorie restriction (ICR) operationalized as the 5:2 diet versus continuous calorie restriction (CCR) on fecal microbiota of 147 overweight or obese adults in a 50-week parallel-arm randomized controlled trial, the HELENA Trial. The primary outcome of the trial was the differential effects of ICR versus CCR on gene expression in subcutaneous adipose tissue. Changes in the gut microbiome, which are the focus of this publication, were defined as exploratory endpoint of the trial. The trial comprised a 12-week intervention period, a 12-week maintenance period, and a final follow-up period of 26 weeks. RESULTS: Both diets resulted in ~5% weight loss. However, except for Lactobacillales being enriched after ICR, post-intervention microbiome composition did not significantly differ between groups. Overall weight loss was associated with significant metabolic improvements, but not with changes in the gut microbiome. Nonetheless, the abundance of the Dorea genus at baseline was moderately predictive of subsequent weight loss (AUROC of 0.74 for distinguishing the highest versus lowest weight loss quartiles). Despite the lack of consistent intervention effects on microbiome composition, significant study group-independent co-variation between gut bacterial families and metabolic biomarkers, anthropometric measures, and dietary composition was detectable. Our analysis in particular revealed associations between insulin sensitivity (HOMA-IR) and Akkermansiaceae, Christensenellaceae, and Tanerellaceae. It also suggests the possibility of a beneficial modulation of the latter two intestinal taxa by a diet high in vegetables and fiber, and low in processed meat. CONCLUSIONS: Overall, our results suggest that the gut microbiome remains stable and highly individual-specific under dietary calorie restriction. TRIAL REGISTRATION: The trial, including the present microbiome component, was prospectively registered at ClinicalTrials.gov NCT02449148 on May 20, 2015.

Blood polyphenol concentrations and differentiated thyroid carcinoma in women from the European Prospective Investigation into Cancer and Nutrition (EPIC) study.

BACKGROUND: Polyphenols are natural compounds with anticarcinogenic properties in cellular and animal models, but epidemiological evidence determining the associations of these compounds with thyroid cancer (TC) is lacking. OBJECTIVES: The aim of this study was to evaluate the relations between blood concentrations of 36 polyphenols and TC risk in EPIC (the European Prospective Investigation into Cancer and Nutrition). METHODS: A nested case-control study was conducted on 273 female cases (210 papillary, 45 follicular, and 18 not otherwise specified TC tumors) and 512 strictly matched controls. Blood polyphenol concentrations were analyzed by HPLC coupled to tandem MS after enzymatic hydrolysis. RESULTS: Using multivariable-adjusted conditional logistic regression models, caffeic acid (ORlog2: 0.55; 95% CI: 0.33, 0.93) and its dehydrogenated metabolite, 3,4-dihydroxyphenylpropionic acid (ORlog2: 0.84; 95% CI: 0.71, 0.99), were inversely associated with differentiated TC risk. Similar results were observed for papillary TC, but not for follicular TC. Ferulic acid was also inversely associated only with papillary TC (ORlog2: 0.68; 95% CI: 0.51, 0.91). However, none of these relations was significant after Bonferroni correction for multiple testing. No association was observed for any of the remaining polyphenols with total differentiated, papillary, or follicular TC. CONCLUSIONS: Blood polyphenol concentrations were mostly not associated with differentiated TC risk in women, although our study raises the possibility that high blood concentrations of caffeic, 3,4-dihydroxyphenylpropionic, and ferulic acids may be related to a lower papillary TC risk.

Changes in Plasma Short-Chain Fatty Acid Levels after Dietary Weight Loss Among Overweight and Obese Adults over 50 Weeks.

Gut microbial-derived short-chain fatty acids (SCFAs) may regulate energy homeostasis and exert anti-carcinogenic, immunomodulatory and anti-inflammatory effects. Smaller trials indicate that dietary weight loss may lead to decreased SCFA production, but findings have been inconclusive. SCFA concentrations were measured by HPLC-MS/MS in plasma samples of 150 overweight or obese adults in a trial initially designed to evaluate the metabolic effects of intermittent (ICR) versus continuous (CCR) calorie restriction (NCT02449148). For the present post hoc analyses, participants were classified by quartiles of weight loss, irrespective of the dietary intervention. Linear mixed models were used to analyze weight-loss-induced changes in SCFA concentrations after 12, 24 and 50 weeks. There were no differential changes in SCFA levels across the initial study arms (ICR versus CCR versus control) after 12 weeks, but acetate concentrations significantly decreased with overall weight loss (mean log-relative change of -0.7 ± 1.8 in the lowest quartile versus. -7.6 ± 2 in the highest, p = 0.026). Concentrations of propionate, butyrate and other SCFAs did not change throughout the study. Our results show that weight-loss, achieved through calorie restriction, may lead to smaller initial decreases in plasma acetate, while plasma SCFAs generally remain remarkably stable over time.

Circulating 27-hydroxycholesterol and breast cancer tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß: results from the EPIC-Heidelberg cohort.

BACKGROUND: Experimental and epidemiological studies demonstrate a role for 27-hydroxycholesterol (27HC) in breast cancer development, though results are conflicting. Cholesterol 27-hydroxylase (CYP27A1) and oxysterol 7-alpha-hydroxylase (CYP7B1) regulate 27HC concentrations, while differential expression of the liver X receptor (LXR) and estrogen receptor beta (ERß) may impact the association between 27HC and breast cancer risk. METHODS: We evaluated correlates of tumor tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß and the association between circulating prediagnostic 27HC concentrations and breast cancer risk by marker expression in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg cohort including 287 breast cancer cases with tumor tissue available. Tumor protein expression was evaluated using immunohistochemistry, and serum 27HC concentrations quantified using liquid chromatography-mass spectrometry. Conditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS: A higher proportion of CYP7B1-positive cases were progesterone receptor (PR)-positive, relative to CYP7B1-negative cases, whereas a higher proportion of ERß-positive cases were Bcl-2 low, relative to ERß-negative cases. No differences in tumor tissue marker positivity were observed by reproductive and lifestyle factors. We observed limited evidence of heterogeneity in associations between circulating 27HC and breast cancer risk by tumor tissue expression of CYP27A1, CYP7B1, LXR-ß, and ERß, with the exception of statistically significant heterogeneity by LXR-ß status in the subgroup of women perimenopausal at blood collection (p = 0.02). CONCLUSION: This exploratory study suggests limited associations between tumor marker status and epidemiologic or breast cancer characteristics. Furthermore, the association between circulating 27HC and breast cancer risk may not vary by tumor expression of CYP27A1, CYP7B1, LXR-ß, or ERß.

Association between lifestyle, dietary, reproductive, and anthropometric factors and circulating 27-hydroxycholesterol in EPIC-Heidelberg.

PURPOSE: Given that 27-hydroxycholesterol (27HC) is the first identified endogenous selective estrogen receptor modulator, the aim of this study was to investigate the extent to which dietary or lifestyle factors impact circulating 27HC concentrations in a large-scale setting. METHODS: This cross-sectional analysis included 1,036 women aged 35-65 years who served as controls in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg breast cancer case-control study. Circulating 27HC was quantified in serum using liquid chromatography/tandem mass spectrometry. Generalized linear models were used to investigate the association between 27HC concentrations and dietary habits, and lifestyle, reproductive, and anthropometric factors. RESULTS: Higher concentrations of 27HC were observed among postmenopausal relative to premenopausal women (geometric mean 200.5 vs. 188.4 nM, p = 0.03), whereas women reporting ever full-term pregnancy had lower concentrations of 27HC relative to never (191.4 vs. 198.6; p = 0.03). Significant trends were observed showing higher concentrations with relatively high levels of physical activity (ptrend = 0.03) and alcohol consumption (ptrend = 0.01), and women currently smoking at blood collection (ptrend < 0.01). Of the investigated dietary factors, starch (ptrend < 0.01) and thiamine (ptrend < 0.01) intakes were inversely associated with 27HC. Circulating lipid concentrations were positively associated with 27HC concentrations (all ptrend < 0.01). No significant associations were found between 27HC and factors including age at blood collection, body mass index, or use of hormone therapy or cholesterol-lowering medications. CONCLUSION: 27HC is of increasing interest for multiple chronic disease pathways. Despite significant associations found between circulating 27HC and dietary habits, reproductive factors, and modifiable lifestyle factors, circulating cholesterol, mostly low-density lipoprotein cholesterol, accounted for the majority of the variability in circulating 27HC.

Effects of Weight-Loss Interventions on Short-Chain Fatty Acid Concentrations in Blood and Feces of Adults: A Systematic Review.

Short-chain fatty acids (SCFAs, mainly acetate, propionate, and butyrate), which are primarily derived from the gut microbiome, may exert anti-inflammatory and immunomodulatory effects, and regulate energy homeostasis. It has been suggested that weight loss may affect SCFA metabolism, but a systematic review of intervention studies is lacking. We aimed to systematically assess the effects of dietary, physical activity-based, and surgical weight-loss interventions among overweight [body mass index (BMI) 25-29.9 kg/m2)] or obese (BMI ≥30 kg/m2) adults (≥18 y) on concentrations of acetate, propionate, butyrate, and total SCFAs in blood, urine, or feces. We conducted a systematic literature search in PubMed, Web of Science, and the Cochrane Central Register of Controlled Trials (CENTRAL) up to April 30, 2018 for randomized and nonrandomized weight-loss trials among overweight or obese adults, in which the concentrations of individual and total SCFAs were assessed. A total of 9 studies consisting of 2 randomized parallel-arm trials, 4 crossover trials, and 3 nonrandomized clinical or surgical trials were included. In the majority of studies, changes in fecal SCFA concentrations were assessed, whereas changes in serum SCFAs were reported from 1 trial. Individual and total SCFA concentrations either remained unchanged or decreased significantly following weight loss. Three of the dietary interventions that resulted in decreased SCFA concentrations were low (≤5% of energy) in total carbohydrates. Most of the studies had a high risk of bias. Decreases in SCFA concentrations may accompany weight loss induced by bariatric surgery or dietary restriction among overweight or obese adults, particularly when carbohydrate intake is reduced. However, findings were inconsistent and based on studies with high to unclear risk of bias, and small sample sizes. Because measurements of fecal SCFAs may not be ideal due to limited sample standardization, well-powered trials with repeated blood measurements of SCFAs are required. This review was registered at PROSPERO as CRD42018088716.

Key Genes of Lipid Metabolism and WNT-Signaling Are Downregulated in Subcutaneous Adipose Tissue with Moderate Weight Loss.

Smaller cross-sectional studies and bariatric surgery trials suggest that weight loss may change the expression of genes in adipose tissue that have been implicated in the development of metabolic diseases, but well-powered intervention trials are lacking. In post hoc analyses of data from a 12-week dietary intervention trial initially designed to compare metabolic effects of intermittent vs. continuous calorie restriction, we analyzed the effects of overall weight loss on the subcutaneous adipose tissue (SAT) transcriptome. Changes in the transcriptome were measured by microarray using SAT samples of 138 overweight or obese individuals (age range: 35⁻65 years, BMI range: 25⁻40, non-smokers, non-diabetics). Participants were grouped post hoc according to the degree of their weight loss by quartiles (average weight loss in quartiles 1 to 4: 0%, -3.2%, -5.9%, and -10.7%). Candidate genes showing differential expression with weight loss according to microarray analyses were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and fold changes (FCs) were calculated to quantify differences in gene expression. A comparison of individuals in the highest vs. the lowest weight loss quartile revealed 681 genes to be differentially expressed (corrected p < 0.05), with 40 showing FCs of at least 0.4. Out of these, expression changes in secreted frizzled-related protein 2 (SFRP2, FC = 0.65, p = 0.006), stearoyl-CoA desaturase (SCD, FC = -1.00, p < 0.001), and hypoxia inducible lipid droplet-associated (HILPDA, FC = -0.45, p = 0.001) with weight loss were confirmed by RT-qPCR. Dietary weight loss induces significant changes in the expression of genes implicated in lipid metabolism (SCD and HILPDA) and WNT-signaling (SFRP2) in SAT.

Circulating 27-Hydroxycholesterol and Breast Cancer Risk: Results From the EPIC-Heidelberg Cohort.

BACKGROUND: 27-hydroxycholesterol (27HC) was the first identified endogenous selective estrogen receptor modulator (SERM); 27HC promoted growth and metastasis in experimental models of estrogen receptor-positive mammary cancer. There are no data on prediagnosis circulating 27HC and breast cancer risk in women. METHODS: We conducted a nested case-control study in the well-characterized Heidelberg, Germany, cohort of the European Investigation into Cancer and Nutrition (EPIC) including 530 incident invasive breast cancer cases, each matched to up to two control participants (n = 1036). Serum 27HC was analyzed by liquid chromatography-mass spectrometry (LC-MS) in blood samples collected at study recruitment. Multivariable conditional logistic regression models were used to quantify the association between circulating 27HC and breast cancer risk overall, by tumor hormone receptor status (ie estrogen and progesterone receptors), and by menopausal status at blood collection. All statistical tests were two-sided. RESULTS: 27HC was not associated with breast cancer risk overall (relative risk [RR]Quartile4vsQuartile1 [Q4vsQ1] = 0.90, 95% confidence interval [CI] = 0.66 to 1.22). The association between 27HC and breast cancer risk differed by menopausal status at blood collection (Phet = .02), but not by age at diagnosis (Phet = .78). Among women who were postmenopausal at blood collection, higher serum 27HC levels were associated with lower breast cancer risk (RRQ4vsQ1 = 0.56, 95% CI = 0.36 to 0.87). We observed no association between 27HC and breast cancer risk (RRQ4vsQ1 = 1.33, 95% CI = 0.75 to 2.38) among women who were premenopausal at blood collection. CONCLUSIONS: In this first prospective study, higher circulating 27HC was associated with lower risk of breast cancer in postmenopausal women. Identification of the first endogenous SERM associated with reduced risk of invasive breast cancer in postmenopausal women may offer novel avenues for breast cancer prevention strategies.

Reproducibility of serum oxysterols and lanosterol among postmenopausal women: Results from EPIC-Heidelberg.

BACKGROUND: Circulating oxysterols have been proposed as biological markers of disease risk. However, within-person reproducibility of circulating oxysterols over time is not well established. METHODS: We evaluated the one-year reproducibility of 11 oxysterols and lanosterol among 30 postmenopausal women with repeat blood samples in the European Prospective Investigation into Cancer and Nutrition (EPIC) - Heidelberg, Germany cohort. Liquid chromatography-mass spectrometry (LC/MS) was performed to quantify serum concentrations of 22R-hydroxycholesterol, 25-hydroxycholesterol, 24S-hydroxycholesterol, 27-hydroxycholesterol, 22S-hydroxycholeterol, 24,25-epoxycholesterol, 5α,6ß-dihydroxycholestanol, 7α-hydroxycholesterol, 5ß,6ß-epoxycholesterol, 5α,6α-epoxycholesterol, 24-dihydrolanosterol, and lanosterol. We evaluated Spearman correlations and intraclass correlation coefficients (ICCs) between quantifiable concentrations measured in repeat samples taken one-year apart to estimate within-person reproducibility. RESULTS: Spearman correlations (ICCs) over one year ranged from 0 (ICC=0.10) for 5ß,6ß-epoxycholesterol and 0.10 (ICC=0.20) for 5α,6α-epoxycholesterol, representing low within-person stability, to 0.81 (ICC=0.75) for 27-hydroxycholesterol and 0.86 (ICC=0.91) for 24S-hydroxycholesterol, representing relatively high within-person stability. Correlations between oxysterols and lanosterol ranged from 0.01 between 24S-hydroxycholesterol and lanosterol to 0.70 between 5α,6α-epoxycholesterol and 5ß,6ß-epoxycholesterol. CONCLUSIONS: Our results demonstrate that for 27-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol and lanosterol, a single serum measurement can reliably estimate average levels over a one-year period. Circulating oxysterols are of increasing interest in epidemiologic studies of chronic disease risk including cancer and cardiovascular disease. Our data suggest that within-person stability of oxysterols differs depending on the individual oxysterol evaluated. We identified four oxysterols and lanosterol as stable over time to inform the use of circulating oxysterols in epidemiologic studies.

Ovarian cancer early detection by circulating CA125 in the context of anti-CA125 autoantibody levels: Results from the EPIC cohort.

CA125 is the best ovarian cancer early detection marker to date; however, sensitivity is limited and complementary markers are required to improve discrimination between ovarian cancer cases and non-cases. Anti-CA125 autoantibodies are observed in circulation. Our objective was to evaluate whether these antibodies (1) can serve as early detection markers, providing evidence of an immune response to a developing tumor, and (2) modify the discriminatory capacity of CA125 by either masking CA125 levels (resulting in lower discrimination) or acting synergistically to improve discrimination between cases and non-cases. We investigated these objectives using a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (EPIC) including 250 cases diagnosed within 4 years of blood collection and up to four matched controls. Circulating CA125 antigen and antibody levels were quantified using an electrochemiluminescence assay. Adjusted areas under the curve (aAUCs) by 2-year lag-time intervals were calculated using conditional logistic regression calibrated toward the absolute risk estimates from a pre-existing epidemiological risk model as an offset-variable. Anti-CA125 levels alone did not discriminate cases from controls. For cases diagnosed <2 years after blood collection, discrimination by CA125 antigen was suggestively higher with higher anti-CA125 levels (aAUC, highest antibody tertile: 0.84 [0.76-0.92]; lowest tertile: 0.76 [0.67-0.86]; phet = 0.06). We provide the first evidence of potentially synergistic discrimination effects of CA125 and anti-CA125 antibodies in ovarian early detection. If these findings are replicated, evaluating CA125 in the context of its antibody may improve ovarian cancer early detection.

Anti-Mullerian hormone and endometrial cancer: a multi-cohort study.

BACKGROUND: The Mullerian ducts are the embryological precursors of the female reproductive tract, including the uterus; anti-Mullerian hormone (AMH) has a key role in the regulation of foetal sexual differentiation. Anti-Mullerian hormone inhibits endometrial tumour growth in experimental models by stimulating apoptosis and cell cycle arrest. To date, there are no prospective epidemiologic data on circulating AMH and endometrial cancer risk. METHODS: We investigated this association among women premenopausal at blood collection in a multicohort study including participants from eight studies located in the United States, Europe, and China. We identified 329 endometrial cancer cases and 339 matched controls. Anti-Mullerian hormone concentrations in blood were quantified using an enzyme-linked immunosorbent assay. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI) across tertiles and for a doubling of AMH concentrations (ORlog2). Subgroup analyses were performed by ages at blood donation and diagnosis, oral contraceptive use, and tumour characteristics. RESULTS: Anti-Mullerian hormone was not associated with the risk of endometrial cancer overall (ORlog2: 1.07 (0.99-1.17)), or with any of the examined subgroups. CONCLUSIONS: Although experimental models implicate AMH in endometrial cancer growth inhibition, our findings do not support a role for circulating AMH in the aetiology of endometrial cancer.

Enterolactone concentrations and prognosis after postmenopausal breast cancer: assessment of effect modification and meta-analysis.

We previously reported that high concentrations of enterolactone, a lignan metabolite, are associated with lower mortality in 1,140 breast cancer patients from Germany. Using an extended set of 2,182 patients aged 50-74 years at diagnosis (2001-2005) and prospectively followed up until 2009, we investigated whether the association with mortality differs by lifestyle factors and tumor characteristics. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using multivariable Cox regression. Potential differential effects by tumor characteristics and lifestyle factors were assessed and a meta-analysis of five studies addressing lignan exposure and breast cancer prognosis was performed to summarize evidence. Median enterolactone concentrations were 17.4 (± 30.5 standard deviation) and 22.9 nmol L(-1) (± 44.8), respectively, for 269 deceased and 1,913 patients still alive. High enterolactone concentrations were significantly associated with lower all-cause mortality (per 10 nmol L(-1) : HR 0.94, 95% CI 0.90-0.98), breast cancer-specific mortality (HR 0.94, 0.89-0.99), and distant disease-free survival (HR 0.94, 0.90-0.98). Associations were found for stage 0-IIIA but not for stage IIIB-IV disease (p(het) = 0.01) and were stronger in patients with BMI <25 kg m(-2) than those with BMI ≥ 25 (p(het) = 0.04). In patients with healthy lifestyle (BMI <25, nonsmoker, physically active), the inverse association with all-cause mortality was still apparent (HR 0.92, 0.85-0.99). The meta-analysis yielded significant associations both for all-cause (HR 0.57, 0.42-0.78) and breast cancer-specific mortality (HR 0.54, 0.39-0.75). Our findings show that high lignan exposure is associated with reduced mortality in breast cancer patients. The inverse association observed in this study cannot be entirely explained by a healthy lifestyle.

Circulating 25-hydroxyvitamin D and postmenopausal breast cancer survival: Influence of tumor characteristics and lifestyle factors?

We previously reported that lower post-diagnostic circulating 25-hydroxyvitamin D [25(OH)D] concentrations were associated with higher risk of overall mortality and distant disease in stage I-IV postmenopausal breast cancer survivors. This association was now re-examined in an extended dataset to investigate potential effect modification by tumor characteristics and lifestyle factors. A prospective cohort study was conducted in Germany including 2,177 incident stage I-IV postmenopausal breast cancer patients aged 50-74 years. Patients were diagnosed between 2001 and 2005 and median follow-up time was 5.3 years. Cox proportional hazards models were stratified by age at diagnosis, study center and season of blood collection and adjusted for other prognostic factors. A meta-analysis of studies on circulating 25(OH)D and mortality in breast cancer patients was performed to summarize evidence. Lower concentrations of 25(OH)D were significantly associated with higher risk of overall mortality [hazard ratio (HR) lowest vs. highest tertile = 1.86; 95% confidence interval (CI): 1.22, 2.82; p-trend = 0.002] and distant disease (HR = 1.76; 95% CI: 1.24, 2.49; p-trend = 0.003) in stage I-IIIa but not in stage IIIb-IV breast cancer patients. No significant interaction by lifestyle factors was observed (all p-interaction > 0.05). The meta-analysis yielded significant associations with overall and breast cancer-specific mortality (lowest vs. highest quantile: HR = 1.52; 95% CI: 1.22, 1.88 and HR = 1.74; 95% CI: 1.23, 2.40, respectively). In conclusion, post-diagnostic circulating 25(OH)D concentrations were associated with overall mortality and distant disease in stage I-IIIa postmenopausal breast cancer patients. This association was not strongly modified by lifestyle factors.

Targeting of the hNC16A collagen domain to dendritic cells induces tolerance to human type XVII collagen.

Antibodies, specific to murine DEC205, can be used to target antigens to dendritic cells. The immunodominant domain of human type XVII collagen, hNC16A, was fused to this antibody (DEC-hNC16A) and was administered as expression plasmid by gene gun transfection with the aim of inducing tolerance to human type XVII collagen in a skin transplantation model. Mice transfected with DEC-hNC16A were challenged with skin grafts from transgenic mice engineered to express human type XVII collagen. Graft survival was either prolonged or grafts were accepted infinitely (33% and 16%, respectively) upon treatment with DEC-hNC16A while 100% of grafts were rejected in untreated controls. Graft acceptance was associated with the absence of a CD4+ infiltrate and a dense CD8+ T-cell infiltrate and was not strictly dependent on antibody production. Our results show that DEC-hNC16A targets dendritic cells in vivo leading to prolonged survival of transgenic skin grafts. This indicates that DEC205-targeting may be used for the induction of tolerance to skin antigens, which would increase the chances of successful skin gene therapy of epidermolysis bullosa patients.

Extracorporeal photophoresis augments function of CD4+CD25+FoxP3+ regulatory T cells by triggering adenosine production.

BACKGROUND: During extracorporeal photophoresis (ECP), peripheral blood mononuclear cells are treated with DNA-intercalating agents and irradiated with ultraviolet light. This procedure exerts immunosuppressive effects, most likely mediated by regulatory T cells (Treg). However, the underlying mechanisms are not clear yet. In our study, we investigated the effect of ECP on frequency and function of Treg in the peripheral blood of patients suffering from graft-versus-host disease. METHODS: Whole blood samples from graft-versus-host disease patients were taken before and after the ECP treatment on 2 consecutive days. Phenotypical analysis of changes in distinct leukocyte subsets within the peripheral blood of patients and healthy controls was performed by means of flow cytometry. Functional analysis of the Treg population after magnetic bead isolation was performed using conventional suppression assays, and adenosine was detected by means of high pressure liquid chromatography and Lanzetta assays. RESULTS: We show that the frequency of CD4/CD25/FoxP3 Treg in the peripheral blood increases after each cycle of ECP and also in the course of treatment. The suppressive capacity of Treg after ECP was increased compared with that of Treg before ECP, although not reaching the suppression levels obtained with Treg from healthy controls. Furthermore, we show that ECP stimulates the CD39-mediated production of adenosine by Treg, which substantially reduces the T-cell proliferation in in vitro suppression assays. CONCLUSION: Our data indicate that ECP stimulates the conversion of ATP to adenosine by the ectonucleotiodase CD39, which acts as a novel soluble immunosuppressive reagent mediating immunosuppression of Treg.

Inhibition of melanoma growth by targeting of antigen to dendritic cells via an anti-DEC-205 single-chain fragment variable molecule.

PURPOSE: Our goal was to target melanoma antigens to the dendritic cell-specific receptor DEC-205. DEC-205 is an antigen receptor expressed on dendritic cells and has been shown to guide antigens to MHC class I and II compartments for processing and presentation to T cells. EXPERIMENTAL DESIGN: The melanoma tumor-associated antigen (TAA), gp100, was fused to the single-chain fragment variable (scFv) specific for DEC-205. The binding capacity of the scFv was tested on lymph node-isolated CD11c+ cells. Mixed lymphocyte reactions were carried out to show an increased proliferative capacity of gp100 antigen-specific CD4 and CD8 T cells. Furthermore the scFv-TAA was used in a therapeutic setting using two different melanoma mouse models. RESULTS: C57Bl/6 mice were injected with scFv-DEC-205-gp100, monoclonal antibody anti-DEC-205, or PBS. Using fluorescence-activated cell sorting, we showed that lymph node CD11c+ dendritic cells stained positive for the binding of the scFv-mDEC-205-gp100 and the anti-DEC-205 monoclonal antibody, whereas the PBS-injected animals were negative. In mixed lymphocyte reactions, bone marrow-derived dendritic cells pulsed with scFv-mDEC-205-gp100 significantly increased proliferation of gp100-specific CD8+ and CD4+ T cells beyond gp100 peptide-pulsed or nonpulsed bone marrow-derived dendritic cells. Finally, in B16/F10 and RET models, a concentration-dependent suppression of tumor growth using scFv-mDEC-205-gp100 (66% reduction of tumor volume), in comparison with gp100 peptide vaccination, was observed. CONCLUSIONS: Our results indicate that the scFv-mDEC-205-gp100 targets TAA to dendritic cells in vivo for presentation on both MHC class I and II molecules. In vivo, this leads to an improved immune response and a decrease in tumor growth rate.

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation.

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.

Tolerogenic dendritic cells and regulatory T cells: a two-way relationship.

At first glance, dendritic cells (DCs) and regulatory T cells (Tregs) do not have much in common. DCs are characterized by their unsurpassed T cell stimulatory capacity, whereas Tregs are marked by the ability to suppress proliferation of effector T cells. However, only mature/activated DCs stimulate T cell proliferation, whereas immature DCs induce Tregs. This provides a means by which peripheral tolerance is maintained: in the absence of inflammation and disease, DCs encounter apoptotic cells and "self" detritus in peripheral tissues. Thus, DCs constantly sample the peripheral environment and, accordingly, the presentation of "self" by these steady state DCs results in induction of suppressive Tregs. Vice versa, Tregs are able to affect DC development, preventing maturation and inducing IL-10, as well as immunosuppressive molecules of the B7-H family, in DCs. Therefore, these novel findings establish a mutual interaction between DCs and Tregs for the upkeep of immunosuppression: immature DCs induce Tregs and inversely Tregs prepare DCs to become immunosuppressive, thereby extending the immunosuppressive function of Tregs. The possible means of cellular interactions as well as the consequences for tolerance and immunity are discussed in this review.