Host and Environmental Factors Influencing Individual Human Cytokine Responses.

Differences in susceptibility to immune-mediated diseases are determined by variability in immune responses. In three studies within the Human Functional Genomics Project, we assessed the effect of environmental and non-genetic host factors of the genetic make-up of the host and of the intestinal microbiome on the cytokine responses in humans. We analyzed the association of these factors with circulating mediators and with six cytokines after stimulation with 19 bacterial, fungal, viral, and non-microbial metabolic stimuli in 534 healthy subjects. In this first study, we show a strong impact of non-genetic host factors (e.g., age and gender) on cytokine production and circulating mediators. Additionally, annual seasonality is found to be an important environmental factor influencing cytokine production. Alpha-1-antitrypsin concentrations partially mediate the seasonality of cytokine responses, whereas the effect of vitamin D levels is limited. The complete dataset has been made publicly available as a comprehensive resource for future studies. PAPERCLIP.

Measuring steroids in hair opens up possibilities to identify congenital adrenal hyperplasia in developing countries.

OBJECTIVE: Patients with congenital adrenal hyperplasia (CAH) in developing countries have limited access to appropriate laboratory facilities for diagnosis and follow-up. The aim of this study is to evaluate steroid measurement in hair as a diagnostic tool to identify and monitor CAH in these patients. DESIGN: A method was developed to measure steroids in hair, the stability of steroids in hair was assessed, and the concentration range in healthy volunteers was determined. Hair samples of patients, before and after starting therapy, were transported at ambient temperature to The Netherlands for analysis. PATIENTS: Twenty-two Indonesian CAH patients and 84 healthy volunteers participated. MEASUREMENTS: Cortisol, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone in hair were measured by liquid chromatography with tandem mass spectrometry. RESULTS: Steroids in hair could be measured and remained stable (<4.9% deviation) for at least 3 weeks at 4°C and 30°C. In each of the untreated patients, hair concentrations of 17OHP (9.43-1135 pmol/g), androstenedione (36.1-432 pmol/g), and testosterone (2.85-69.2 pmol/g) were all above the upper limit of the corresponding range in healthy volunteers; 5.5 pmol/g, 13 pmol/g, and 1.8 pmol/g, respectively. After starting glucocorticoid treatment, the steroid concentrations in the hair of CAH patients decreased significantly for androstenedione (73%) and testosterone (59%) after 6 months. CONCLUSIONS: CAH could be confirmed in Indonesian patients based on the concentration of 17OHP, androstenedione, and testosterone in hair, and a treatment effect was observed. These findings open up opportunities to diagnose and/or monitor CAH in developing countries with a simple noninvasive technique.

Psychological impact over time of women with pregnancy loss due to gestational trophoblastic disease compared with miscarriage.

OBJECTIVES: Pregnancy loss, occurring after miscarriage or after gestational trophoblastic disease, has a psychological impact. Besides pregnancy loss, women diagnosed with gestational trophoblastic disease have to deal with a prolonged period of follow-up and potential advice to postpone a future pregnancy. We studied the severity and course of the psychological impact after gestational trophoblastic disease and miscarriage, to identify whether women with gestational trophoblastic disease need different psychological care. METHODS: A prospective multicenter study using online questionnaires was performed. Women diagnosed with gestational trophoblastic disease or miscarriage received the following questionnaires directly after diagnosis, and after 6, 6, and 12 months: a self-report questionnaire, the Hospital Anxiety and Depression Scale (HADS), the Impact of Event Scale, and the Reproductive Concerns Scale. RESULTS: 74 women with gestational trophoblastic disease and 76 women with miscarriage were included. At baseline, the proportion of women scoring above the cut-off level for the anxiety subscale of the HADS and for the Impact of Event Scale was significantly higher for women with gestational trophoblastic disease than for women after miscarriage (43.2% vs 28.9%, p=0.02 and 87.8% vs 78.9%, p=0.03, respectively). During follow-up, the differences between both groups vanished and only the Impact of Event Scale after 12 months remained significantly different between women with gestational trophoblastic disease and women after miscarriage (62.7% vs 37.3%, p=0.005). All outcomes, except the Reproductive Concerns Scale, showed a significant decline. However, in women who scored above the cut-off level on the HADS-total or Impact of Event Scale at baseline, and women with psychological or psychiatric history, significant higher scores persisted. CONCLUSION: Although women with gestational trophoblastic disease at baseline had more anxiety and distress than women after miscarriage, no significant differences were seen using the HADS-total after 12 months. Using the HADS or Impact of Event Scale directly after pregnancy loss is helpful to identify women at risk of remaining psychological symptoms to provide them with extra psychological support.

Measurement of hCG in women with Gestational Trophoblastic Disease.

OBJECTIVES: The objective of this study was to collect information on human Chorionic Gonadotrophin (hCG) laboratory testing and reporting in women with Gestational Trophoblastic Disease (GTD), to assess the associated challenges, and to offer perspectives on hCG testing harmonisation. DESIGN: Information was collected from laboratories by electronic survey (Survey Monkey®) using a questionnaire designed by members of the European Organisation for the Treatment of Trophoblastic Disease (EOTTD) hCG Working Party. PARTICIPANTS: The questionnaire was distributed by the EOTTD board to member laboratories and their associated scientists who work within the GTD field. SETTING: The questionnaire was distributed and accessed via an online platform. METHODS: The questionnaire consisted of 5 main sections. These included methods used for hCG testing, quality procedures, reporting of results, laboratory operational aspects, and non-GTD testing capability. In addition to reporting these survey results, examples of case scenarios which illustrate the difficulties faced by laboratories providing hCG measurement for GTD patient management were described. The benefits and challenges of using centralised versus non-centralised hCG testing were discussed alongside the utilisation of regression curves for management of GTD patients. RESULTS: Information from the survey was collated and presented for each section and showed huge variability in responses across laboratories even for those using the same hCG testing platforms. An educational example was presented, highlighting the consequence of using inappropriate hCG assays on clinical patient management (Educational Example A), along with an example of biotin interference (Educational Example B) and an example of high-dose hook effect (Educational Example C), demonstrating the importance of knowing the limitations of hCG tests. The merits of centralised versus non-centralised hCG testing and use of hCG regression curves to aid patient management were discussed. LIMITATIONS: To ensure the survey was completed by laboratories providing hCG testing for GTD management, the questionnaire was distributed by the EOTTD board. It was assumed the EOTTD board held the correct laboratory contact, and that the questionnaire was completed by a scientist with in-depth knowledge of laboratory procedures. CONCLUSIONS: The hCG survey highlighted a lack of harmonisation of hCG testing across laboratories. Healthcare professionals involved in the management of women with GTD should be aware of this limitation. Further work is needed to ensure an appropriate quality assured laboratory service is available for hCG monitoring in women with GTD.

Evaluation of a Web-Based Intervention for Patients with Gestational Trophoblastic Disease: A Randomized Controlled Trial.

OBJECTIVE: Gestational trophoblastic diseases (GTD) comprise a group of rare diseases originating from the trophoblast affecting women of childbearing age. Providing optimal information to patients with a rare disease is challenging because of the small number of patients and limited clinical expertise of many healthcare professionals. Both knowledge and lack of knowledge in patients may influence illness perception. We investigated whether a web-based interactive intervention influences illness perception and knowledge in women with GTD. DESIGN: This was a multicenter randomized control trial conducted at general and academic hospitals in the Netherlands, including newly diagnosed GTD patients between 2017 and 2019. METHODS: Sixty-nine patients were randomized between direct access or postponed access to an online tool on GTD and received online questionnaires about illness perception, knowledge, and anxiety. The main outcome measures were illness perception (primary outcome measure) and knowledge (secondary outcome measure). RESULTS: Patients using the online tool were satisfied with the information from the tool (92%). Although they had a higher level of knowledge compared to the control group (p = 0.006), illness perception did not change. Also, no differences in levels of anxiety, depression, or distress were observed between the groups. LIMITATIONS: Participants had access to other information sources and many searched other websites. It is unknown what kind of websites were visited and when. It is unknown if the increased knowledge levels and low levels of distress will sustain over time as no long term follow-up took place. Healthcare professionals were not interviewed on how they experienced the consultation before and after using the tool by the patients. CONCLUSIONS: The online tool did not change illness perception but was shown to be valuable for newly diagnosed GTD patients to gain knowledge. The improvement in knowledge after digital education indicates that this tool can be used as an effective method of supporting GTD patients' informational needs without causing extra distress. TWEETABLE ABSTRACT: A web-based tool for trophoblastic disease does not change illness perception of patients but is valuable to gain knowledge.

The circular RNome of primary breast cancer.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative (R < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of CNOT2, CREBBP, and RERE in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of circCNOT2 was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, CNOT2 levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that circCNOT2 is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.

Diurnal salivary androstenedione and 17-hydroxyprogesterone levels in healthy volunteers for monitoring treatment efficacy of patients with congenital adrenal hyperplasia.

OBJECTIVE: Treatment of congenital adrenal hyperplasia (CAH) patients with glucocorticoids is often challenging since there is a delicate balance between over- and undertreatment. Treatment can be monitored noninvasively by measuring salivary androstenedione (A4) and 17-hydroxyprogesterone (17-OHP). Optimal treatment monitoring requires the establishment of reference values in saliva. DESIGN: A descriptive study. PATIENTS: For this study saliva of 255 healthy paediatric and adult volunteers with an age range of 4-75 years old was used. MEASUREMENTS: We developed a sensitive liquid chromatography-tandem mass spectrometry method, assessed salivary A4 and 17-OHP stability, and measured A4 and 17-OHP concentrations in saliva collected in the morning, afternoon, and evening. RESULTS: We quantified A4 and 17-OHP concentrations in the morning, afternoon, and evening and demonstrated that there is a significant rhythm with the highest levels in the morning and decreasing levels over the day. A4 and 17-OHP concentrations display an age-dependent pattern. These steroids remain stable in saliva at ambient temperature for up to 5 days. CONCLUSIONS: Good stability of the steroids in saliva enables saliva collection by the patient at home. Since salivary A4 and 17-OHP display a diurnal rhythm and age-dependent pattern, we established reference values for both children and adults at three time points during the day. These reference values support treatment monitoring of children and adults with CAH.

PD-L1 in gestational trophoblastic disease: an antibody evaluation.

INTRODUCTION: Treatment with antibodies directed against programed-cell death ligand 1 (PD-L1) is a novel therapy for patients with gestational trophoblastic disease. Assessment of PD-L1 expression in tumor tissue is commonly used to identify patients who might benefit from anti-PD-L1 treatment. Multiple antibodies are available to detect PD-L1-expressing cells, and percentages of PD-L1-expressing cells in samples of patients with gestational trophoblastic disease indicated by these antibodies differ substantially. This raises the question which PD-L1 antibody best reflects PD-L1 expression to select patients for treatment. MATERIAL AND METHODS: Seven commercially available antibodies for PD-L1 staining (E1L3N, 73-10, 22C3, CAL10, SP142, 28-8, SP263) were validated on Chinese hamster ovarian (CHO) cells transfected with PD-L1, PD-L2, wildtype CHO cells and tonsil tissue. Next, four complete hydatidiform moles and four choriocarcinomas were stained. Samples were independently assessed by two pathologists. RESULTS: All seven antibodies showed membranous staining in the PD-L1-transfected CHO cells. E1L3N and 22C3 scored the highest percentages of PD-L1-positive cells (70%-90% and 60%-70%, respectively). E1L3N stained the cytoplasm of non-transfected CHO cells and was excluded from analysis. The remaining six antibodies predominantly stained syncytiotrophoblast cells of both complete hydatidiform moles and choriocarcinomas. The percentage of PD-L1-stained trophoblast cells and staining intensity varied substantially per used PD-L1 antibody and between complete hydatidiform moles and choriocarcinomas. Agreement between pathologists was best with 22C3 (intraclass correlation coefficient 0.94-0.96). CONCLUSIONS: Based on staining results of the CHO cells, gestational trophoblastic disease samples and intraclass correlation coefficient, 22C3 seems the most suitable for adequate detection of PD-L1-expressing trophoblast cells. All antibodies detected PD-L1-expressing cells in the gestational trophoblastic disease samples, though with great variability, hampering comparison of results between studies in this rare disease and emphasizing the need for uniformity in detecting PD-L1-expressing cells.

Harmonization of LC-MS/MS Measurements of Plasma Free Normetanephrine, Metanephrine, and 3-Methoxytyramine.

BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.

Early prediction of post-molar gestational trophoblastic neoplasia and resistance to methotrexate, based on a single serum human chorionic gonadotropin measurement.

BACKGROUND: Clinicians are unable to provide individualized counseling regarding risk of progression for patients with a complete hydatidiform mole (CHM). We developed nomograms enabling early prediction of post-molar gestational trophoblastic neoplasia (GTN) and resistance to methotrexate (MTX) based on a single serum human chorion gonadotropin (hCG) measurement. METHODS: We generated two nomograms with logistic regression: to predict post-molar GTN, and MTX resistance. For patients with high probability to progress to post-molar GTN or MTX resistance, we determined hCG cut-offs at 97.5% specificity to select patients for additional- or adjustments in current treatment. RESULTS: The nomograms had a good to excellent ability to distinguish either between patients with uneventful hCG regression versus progression to post molar GTN, or between patients cured by MTX versus patients in whom resistance would occur. At 97.5% specificity, we identified 66% (95%CI 56-75) of the 149 patients who would progress to post-molar GTN, four weeks after initial curettage. For patients treated with MTX, we identified 55% (95%CI 23-83) of the 43 patients who would become resistant, preceding their third course at 97.5% specificity. CONCLUSION: The nomograms and cut-off levels can be used to assist in counseling for patients diagnosed with CHM.

Immune cell composition in the endometrium of patients with a complete molar pregnancy: Effects on outcome.

OBJECTIVE: In 15% of patients with complete hydatidiform mole (CHM), disease progresses to post-molar gestational trophoblastic neoplasia (GTN) after curettage. Tumor infiltrating lymphocytes (TILs) are essential in overcoming disease in many tumors. Infiltrating lymphocyte composition and density may influence trophoblast regression and development of post-molar GTN. We analyzed immune cell composition and density in curettaged endometrium of patients with CHM which spontaneously regressed, and of patients with CHM which progressed to post-molar GTN. METHODS: Sixteen patients with CHM and spontaneous regression, and 16 patients with CHM which progressed to post-molar GTN were selected. Immune cell composition and density of natural killer (NK) cells, natural killer T (NKT)-like cells, Cytotoxic T cells, T-Regulatory and T-Helper cells, were determined by multiplex immunohistochemistry (mIHC). RESULTS: Curettaged endometrium of patients with CHM and spontaneous regression contained a slightly higher number of immune cells compared to patients with CHM which progressed to post-molar GTN. NKT-like cell density was significantly higher in patients with spontaneous regression compared to patients with CHM which progressed to post-molar GTN (483 ± 296 vs.295 ± 143 (mean ± SD), p = 0.03) respectively. NKT-like cell density in the spontaneous regression group was split in 'high' and 'low' (i.e. above and below the median number of NKT-like cells). In patients with high NKT-like cell density, hCG normalized earlier than in patients with low NKT-like cell density (9.5 weeks, (range 3.7-14) vs. 12.9 weeks, (range 8.6-17.9), p = 0.05). CONCLUSION: A high number of NKT-like cells in the endometrium of CHMs may contribute to spontaneous regression of molar trophoblast cells.

A tutorial on dynamic risk prediction of a binary outcome based on a longitudinal biomarker.

Dynamic risk predictions based on all available information are useful in timely identification of high-risk patients. However, in contrast with time to event outcomes, there is still a lack of studies that clearly demonstrate how to obtain and update predictions for a future binary outcome using a repeatedly measured biomarker. The aim of this study is to give an illustrative overview of four approaches to obtain such predictions: likelihood based two-stage method (2SMLE), likelihood based joint model (JMMLE), Bayesian two-stage method (2SB), and Bayesian joint model (JMB). We applied the approaches to provide weekly updated predictions of post-molar gestational trophoblastic neoplasia (GTN) based on age and repeated measurements of human chorionic gonadotropin (hCG). Discrimination and calibration measures were used to compare the accuracy of the weekly predictions. Internal validation of the models was conducted using bootstrapping. The four approaches resulted in the same predictive and discriminative performance in predicting GTN. A simulation study showed that the joint models outperform the two-stage methods when we increase the within- and the between-patients variability of the biomarker. The applicability of these models to produce dynamic predictions has been illustrated through a comprehensive explanation and accompanying syntax (R and SAS® ).

The macrophage migration inhibitory factor pathway in human B cells is tightly controlled and dysregulated in multiple sclerosis.

In MS, B cells survive peripheral tolerance checkpoints to mediate local inflammation, but the underlying molecular mechanisms are relatively underexplored. In mice, the MIF pathway controls B-cell development and the induction of EAE. Here, we found that MIF and MIF receptor CD74 are downregulated, while MIF receptor CXCR4 is upregulated in B cells from early onset MS patients. B cells were identified as the main immune subset in blood expressing MIF. Blocking of MIF and CD74 signaling in B cells triggered CXCR4 expression, and vice versa, with separate effects on their proinflammatory activity, proliferation, and sensitivity to Fas-mediated apoptosis. This study reveals a new reciprocal negative regulation loop between CD74 and CXCR4 in human B cells. The disturbance of this loop during MS onset provides further insights into how pathogenic B cells survive peripheral tolerance checkpoints to mediate disease activity in MS.

High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis.

HE4 is superior to CA125 in the detection of recurrent disease in high-risk endometrial cancer patients.

OBJECTIVE: To date, biomarkers are not routinely used in endometrial cancer diagnosis, prognosis, and follow-up. The purpose of this study was to evaluate whether serum HE4 was related to clinicopathological risk factors and outcome. Second, the role of serum HE4 and CA125 was assessed as indicator for recurrent disease during follow-up. METHODS: A total of 174 patients with endometrial cancer between 1999 and 2009 were selected for this retrospective study. Serum HE4 and CA125 were analyzed at primary diagnosis, during follow-up, and at the time of recurrence. Correlations with clinicopathological factors were studied by univariate and multivariate survival analyses. Lead time was calculated in order to determine which serum marker was elevated prior to clinical detection of recurrent disease. RESULTS: Serum levels of HE4 and CA125 were significantly associated with high tumor grade, myometrial invasion, lymph node involvement, and advanced stage (p < 0.01). HE4 was an independent prognostic factor for reduced disease-free survival and overall survival with hazard ratios of 2.96 (95% confidence interval: 1.18-7.99) and 3.27 (95% confidence interval: 1.18-9.02), respectively. At recurrence, 75% of the patients had an elevated HE4 compared to 54% with an elevated CA125. HE4 levels were more frequently elevated in patients with distant metastasis compared to local recurrences, 67% and 37%, respectively. Serum HE4 detected a recurrence with a median of 126 days earlier than clinical confirmation. CONCLUSION: Elevated serum HE4 is an independent risk factor for reduced disease-free survival and overall survival. HE4 seems to be superior to CA125 in the detection of recurrent disease during follow-up, mainly in high-risk endometrial cancer patients who are more prone to distant metastasis.

Tissue kallikrein-related peptidase 4 (KLK4), a novel biomarker in triple-negative breast cancer.

Triple-negative breast cancer (TNBC), lacking the steroid hormone receptors ER and PR and the oncoprotein HER2, is characterized by its aggressive pattern and insensitivity to endocrine and HER2-directed therapy. Human kallikrein-related peptidases KLK1-15 provide a rich source of serine protease-type biomarkers associated with tumor growth and cancer progression for a variety of malignant diseases. In this study, recombinant KLK4 protein was generated and affinity-purified KLK4-directed polyclonal antibody pAb587 established to allow localization of KLK4 protein expression in tumor cell lines and archived formalin-fixed, paraffin-embedded TNBC tumor tissue specimens. For this, KLK4 protein expression was assessed by immunohistochemistry in primary tumor tissue sections (tissue microarrays) of 188 TNBC patients, mainly treated with anthracycline- or CMF-based polychemotherapy. KLK4 protein is localized in the cytoplasm of tumor and stroma cells. In this patient cohort, elevated stroma cell KLK4 expression, but not tumor cell KLK4 expression, is predictive for poor disease-free survival by univariate analysis (hazard ratio: 2.26, p=0.001) and multivariable analysis (hazard ratio: 2.12, p<0.01). Likewise, univariate analysis revealed a trend for statistical significance of elevated KLK4 stroma cell expression for overall survival of TNBC patients as well.

Serum Human Chorionic Gonadotropin Normogram for the Detection of Gestational Trophoblastic Neoplasia.

OBJECTIVE: The aim of this study was to develop a serum human chorionic gonadotropin (hCG) normogram for both uneventful complete and partial hydatidiform moles in the first-trimester ultrasound era. METHODS: An hCG normogram for both complete and partial hydatidiform moles was constructed, based on 639 patients with uneventful serum hCG regression after evacuation between 1990 and 2014. Serum hCG was measured by an in-house-developed radioimmunoassay, detecting both intact hCG and free ß-subunit. It has been in use for all serum measurements sent to the Dutch Central Registry for Hydatidiform Moles since 1977. RESULTS: Since introduction of routine first-trimester ultrasonography, lower pre-evacuation and follow-up serum hCG concentrations were observed. When compared with complete hydatidiform moles, patients with a partial hydatidiform mole had significantly lower pre-evacuation serum hCG concentration (median, 4400 and 875 ng/mL, respectively; P < 0.001) and earlier hCG normalization (median, 7 and 6 weeks, respectively; P < 0.001) but higher gestational age (mean, 11.5 and 13.0 weeks, respectively; P < 0.001). For both complete and partial hydatidiform moles, 95% of patients reached normal serum hCG concentrations within 14 weeks after evacuation. CONCLUSIONS: A normogram for the detection of gestational trophoblastic neoplasia was developed for complete and partial hydatidiform moles. Although interesting from a scientific perspective, the small divergence in hCG regression between complete and partial hydatidiform moles will be of little importance in clinical practice, as actual differences in regression will encompass only days. To promote clarity and unity in daily practice, we therefore propose a combined normogram to be used as a reference guideline for follow-up after evacuation of a hydatidiform mole. This normogram will be compliant with patients in today's clinical practice.

Voluntary activation of the sympathetic nervous system and attenuation of the innate immune response in humans.

Excessive or persistent proinflammatory cytokine production plays a central role in autoimmune diseases. Acute activation of the sympathetic nervous system attenuates the innate immune response. However, both the autonomic nervous system and innate immune system are regarded as systems that cannot be voluntarily influenced. Herein, we evaluated the effects of a training program on the autonomic nervous system and innate immune response. Healthy volunteers were randomized to either the intervention (n = 12) or control group (n = 12). Subjects in the intervention group were trained for 10 d in meditation (third eye meditation), breathing techniques (i.a., cyclic hyperventilation followed by breath retention), and exposure to cold (i.a., immersions in ice cold water). The control group was not trained. Subsequently, all subjects underwent experimental endotoxemia (i.v. administration of 2 ng/kg Escherichia coli endotoxin). In the intervention group, practicing the learned techniques resulted in intermittent respiratory alkalosis and hypoxia resulting in profoundly increased plasma epinephrine levels. In the intervention group, plasma levels of the anti-inflammatory cytokine IL-10 increased more rapidly after endotoxin administration, correlated strongly with preceding epinephrine levels, and were higher. Levels of proinflammatory mediators TNF-α, IL-6, and IL-8 were lower in the intervention group and correlated negatively with IL-10 levels. Finally, flu-like symptoms were lower in the intervention group. In conclusion, we demonstrate that voluntary activation of the sympathetic nervous system results in epinephrine release and subsequent suppression of the innate immune response in humans in vivo. These results could have important implications for the treatment of conditions associated with excessive or persistent inflammation, such as autoimmune diseases.

Identification of novel genetic Loci associated with thyroid peroxidase antibodies and clinical thyroid disease.

Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis), as well as autoimmune hyperthyroidism (Graves' disease). As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives) and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P<5×10(-8)) were detected at TPO-rs11675434, ATXN2-rs653178, and BACH2-rs10944479 for TPOAb-positivity, and at TPO-rs11675434, MAGI3-rs1230666, and KALRN-rs2010099 for TPOAb levels. Individual and combined effects (genetic risk scores) of these variants on (subclinical) hypo- and hyperthyroidism, goiter and thyroid cancer were studied. Individuals with a high genetic risk score had, besides an increased risk of TPOAb-positivity (OR: 2.18, 95% CI 1.68-2.81, P = 8.1×10(-8)), a higher risk of increased thyroid-stimulating hormone levels (OR: 1.51, 95% CI 1.26-1.82, P = 2.9×10(-6)), as well as a decreased risk of goiter (OR: 0.77, 95% CI 0.66-0.89, P = 6.5×10(-4)). The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, which was replicated in an independent cohort of patients with Graves' disease (OR: 1.37, 95% CI 1.22-1.54, P = 1.2×10(-7) and OR: 1.25, 95% CI 1.12-1.39, P = 6.2×10(-5)). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18-2.10, P = 1.9×10(-3)). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb-positives are particularly at risk of developing clinical thyroid dysfunction.