Evaluating Stacked Methylation Markers for Blood-Based Multicancer Detection.

The ability to detect several types of cancer using a non-invasive, blood-based test holds the potential to revolutionize oncology screening. We mined tumor methylation array data from the Cancer Genome Atlas (TCGA) covering 14 cancer types and identified two novel, broadly-occurring methylation markers at TLX1 and GALR1. To evaluate their performance as a generalized blood-based screening approach, along with our previously reported methylation biomarker, ZNF154, we rigorously assessed each marker individually or combined. Utilizing TCGA methylation data and applying logistic regression models within each individual cancer type, we found that the three-marker combination significantly increased the average area under the ROC curve (AUC) across the 14 tumor types compared to single markers (p = 1.158 × 10-10; Friedman test). Furthermore, we simulated dilutions of tumor DNA into healthy blood cell DNA and demonstrated increased AUC of combined markers across all dilution levels. Finally, we evaluated assay performance in bisulfite sequenced DNA from patient tumors and plasma, including early-stage samples. When combining all three markers, the assay correctly identified nine out of nine lung cancer plasma samples. In patient plasma from hepatocellular carcinoma, ZNF154 alone yielded the highest combined sensitivity and specificity values averaging 68% and 72%, whereas multiple markers could achieve higher sensitivity or specificity, but not both. Altogether, this study presents a comprehensive pipeline for the identification, testing, and validation of multi-cancer methylation biomarkers with a considerable potential for detecting a broad range of cancer types in patient blood samples.

The TRH test provides valuable information in the diagnosis of central hypothyroidism in patients with known pituitary disease and low T4 levels.

Objective: To evaluate the value of the thyrotropin-releasing hormone (TRH) test in the diagnosis of central hypothyroidism (CH) in patients with pituitary disease. Methods: Systematic evaluation of 359 TRH tests in patients with pituitary disease including measurements of thyroxine (T4), TBG-corrected T4 (T4corr), baseline TSH (TSH0) and relative or absolute TSH increase (TSHfold, TSHabsolute). Results: Patients diagnosed with CH (n=39) show comparable TSH0 (p-value 0.824) but lower T4corr (p-value <0.001) and lower TSH increase (p-value <0.001) compared to patients without CH. In 54% (42 of 78 cases) of patients with low T4corr, the CH diagnosis was rejected based on a high TSHfold. In these cases, a spontaneous increase and mean normalization in T4corr (from 62 to 73 nmol/L, p-value <0.001) was observed during the follow-up period (7.6 ± 5.0 years). Three of the 42 patients (7%) were started on replacement therapy due to spontaneous deterioration of thyroid function after 2.8 years. Patients diagnosed with CH reported significantly more symptoms of hypothyroidism (p-value 0.005), although, symptoms were reported in most patients with pituitary disease. The TRH test did not provide clinical relevant information in patients with normal T4 or patients awaiting pituitary surgery (78%, 281 of 359). There were only mild and reversible adverse effects related to the TRH test except for possibly one case (0.3%) experiencing a pituitary apoplexy. Conclusion: The TRH test could be reserved to patients with pituitary disease, low T4 levels without convincing signs of CH. Approximately 50% of patients with a slightly decreased T4 were considered to have normal pituitary thyroid function based on the TRH test results.

Atypical presentation of severe pre-eclampsia with sinus bradycardia and chest pain.

This is a case report of an observation of bradycardia and inverted T-waves anteroseptally on the electrocardiogram along with cardiac symptoms, in a previously healthy 35-year-old woman with post-partum pre-eclampsia. Initially, she had no hypertension or proteinuria, which delayed the time of diagnosis. A possible explanation of bradycardia is a baroreceptor-mediated response to hypertension and hypervolaemia. The changes on the electrocardiogram can be explained by pectus excavatum, an enlarged uterus and endothelial dysfunction. One should always consider peri-partum as well as post-partum pre-eclampsia.

Detection of VAR2CSA-Captured Colorectal Cancer Cells from Blood Samples by Real-Time Reverse Transcription PCR.

Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT-qPCR using proprietary Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody-based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low-level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near-perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike-in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT-qPCR assay after rVAR2 capture (p < 0.01 for both primer targets, Mann-Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.

The geropathology of organ-specific aging.

Aging is a complex multidimensional process of progressive decline affecting multiple organ systems by a number of processes that are still not well understood. While many studies have focused on the approach of studying aging across multiple organs, assessment of the contribution of individual organs to overall aging processes is under appreciated. The ability to study and compare organs in the context of organismal aging has been documented recently using a geropathology grading platform in laboratory mice. This concept consists of identifying and grading age-related histologic lesions within organs to generate a quantitative lesion score for each organ, which is representative of the presence and degree of organ-related pathology, and can be compared to scores from other organs examined. This geropathology approach provides a powerful tool to elucidate the basic mechanisms of aging in multiple organs, as well as the response of organs to therapeutic interventions. Furthermore, ongoing work with the concept has expanded and adapted the geropathology grading system to other preclinical animal model species that are commonly used to understand disease associated phenotypes in aging humans, ultimately adding to the utility of the concept.

The Role of Proteoglycans in Cancer Metastasis and Circulating Tumor Cell Analysis.

Circulating tumor cells (CTCs) are accessible by liquid biopsies via an easy blood draw. They represent not only the primary tumor site, but also potential metastatic lesions, and could thus be an attractive supplement for cancer diagnostics. However, the analysis of rare CTCs in billions of normal blood cells is still technically challenging and novel specific CTC markers are needed. The formation of metastasis is a complex process supported by numerous molecular alterations, and thus novel CTC markers might be found by focusing on this process. One example of this is specific changes in the cancer cell glycocalyx, which is a network on the cell surface composed of carbohydrate structures. Proteoglycans are important glycocalyx components and consist of a protein core and covalently attached long glycosaminoglycan chains. A few CTC assays have already utilized proteoglycans for both enrichment and analysis of CTCs. Nonetheless, the biological function of proteoglycans on clinical CTCs has not been studied in detail so far. Therefore, the present review describes proteoglycan functions during the metastatic cascade to highlight their importance to CTCs. We also outline current approaches for CTC assays based on targeting proteoglycans by their protein cores or their glycosaminoglycan chains. Lastly, we briefly discuss important technical aspects, which should be considered for studying proteoglycans.

Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells.

Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval.

Capture and Detection of Circulating Glioma Cells Using the Recombinant VAR2CSA Malaria Protein.

Diffuse gliomas are the most common primary malignant brain tumor. Although extracranial metastases are rarely observed, recent studies have shown the presence of circulating tumor cells (CTCs) in the blood of glioma patients, confirming that a subset of tumor cells are capable of entering the circulation. The isolation and characterization of CTCs could provide a non-invasive method for repeated analysis of the mutational and phenotypic state of the tumor during the course of disease. However, the efficient detection of glioma CTCs has proven to be challenging due to the lack of consistently expressed tumor markers and high inter- and intra-tumor heterogeneity. Thus, for this field to progress, an omnipresent but specific marker of glioma CTCs is required. In this article, we demonstrate how the recombinant malaria VAR2CSA protein (rVAR2) can be used for the capture and detection of glioma cell lines that are spiked into blood through binding to a cancer-specific oncofetal chondroitin sulfate (ofCS). When using rVAR2 pull-down from glioma cells, we identified a panel of proteoglycans, known to be essential for glioma progression. Finally, the clinical feasibility of this work is supported by the rVAR2-based isolation and detection of CTCs from glioma patient blood samples, which highlights ofCS as a potential clinical target for CTC isolation.

Fighting Cancer Using an Oncofetal Glycosaminoglycan-Binding Protein from Malaria Parasites.

Malaria research has led to the discovery of oncofetal chondroitin sulfate, which appears to be shared between placental trophoblasts and cancer cells and can be detected by the evolutionary refined malaria protein VAR2CSA. Interestingly, using recombinant VAR2CSA to target oncofetal chondroitin sulfate shows promise for novel cancer diagnostics and therapeutics.

The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner.

Isolation of metastatic circulating tumor cells (CTCs) from cancer patients is of high value for disease monitoring and molecular characterization. Despite the development of many new CTC isolation platforms in the last decade, their isolation and detection has remained a challenge due to the lack of specific and sensitive markers. In this feasibility study, we present a method for CTC isolation based on the specific binding of the malaria rVAR2 protein to oncofetal chondroitin sulfate (ofCS). We show that rVAR2 efficiently captures CTCs from hepatic, lung, pancreatic, and prostate carcinoma patients with minimal contamination of peripheral blood mononuclear cells. Expression of ofCS is present on epithelial and mesenchymal cancer cells and is equally preserved during epithelial-mesenchymal transition of cancer cells. In 25 stage I-IV prostate cancer patient samples, CTC enumeration significantly correlates with disease stage. Lastly, rVAR2 targets a larger and more diverse population of CTCs compared to anti-EpCAM strategies.

[A rare type of severe obesity in children and adolescents].

A four-year-old girl was referred to a paediatric department with low height, obesity and hypothyroidism. Her paraclinical tests were characteristic with elevated P-parathyroid hormone concentration, hypothyroidism, growth hormone deficiency, abnormal phenotype with brachydactyly, tooth problems and mental retardation, which led to a suspicion of Albright's hereditary osteodystrophy (AHO). The diagnosis was verified by molecular genetic testing. Less than 1% of children with obesity have an endocrine disorder, and AHO is one of them.

Increasing time interval and decreasing allergen dose interval improves ex vivo desensitization of human blood basophils.

BACKGROUND: Desensitization is a method for inducing temporary tolerance to allergen. The mechanism underlying desensitization is yet to be established. METHODS: Basophil granulocytes in whole blood from grass pollen allergic subjects were desensitized ex vivo by sequential addition of increasing allergen concentrations. At each step basophil activation (CD193 + CD63+ ) was monitored with and without (background activation) allergen challenge at optimal concentration. The sequential desensitization protocol was compared to two single-dose desensitization protocols with threshold and subthreshold allergen concentrations. Incubation intervals and allergen concentrations were varied in order to optimise the protocol. RESULTS: Sequential desensitization effectively reduced basophil response. The single-dose subthreshold protocol and single-dose threshold protocol did not reduce basophil activation with optimal allergen challenge from a mean 57.1 (95% CI: 32.7 - 81.5) to 50.4% (95% CI: 16.3 - 84.4; n = 5; P = 0.43) and 45.0% (95% CI: 23.1 - 66.9; P = 0.14) respectively, while the sequential desensitization protocol reduced activation to a mean 37.2% (95% CI: 16.3 - 58.1; P = 0.018). Reducing incubation time from 10 to 5 minutes increased mean background activation from 22.4 (95% CI: 11.7 - 33.1) to 30.0% (95% CI: 19.7 - 40.3; n = 5; P = 0.026). Increasing time intervals from 10 to 20 minutes reduced background activation from 30.9 (95% CI: 22.8 - 39.0) to 21.9% (95% CI: 16.0 - 27.7; n = 5; P = 0.020). Increasing allergen concentration intervals from 2-fold to 5- and 10-fold did not have significant effect on basophil activation. CONCLUSIONS: Sequential desensitization ex vivo effectively attenuates the basophil response to allergen. Increasing the time spent at each step improves desensitization. This protocol could be valuable for investigation the mechanism of desensitization. © 2016 International Clinical Cytometry Society.

The role of matrix metalloproteinases in muscle and adipose tissue development and meat quality: A review.

Matrix metalloproteinases (MMPs) are a group of enzymes that degrade extracellular matrix components but are also important signaling molecules that regulate many biological processes including muscle, adipose and connective tissue development. Most recently it has been discovered that MMPs act as intracellular signaling molecules inducing gene expression and altering related proteins in the nucleus. Several single nucleotide polymorphisms of MMPs and their inhibitors are known to exist and most of the research on MMPs to date has focused on their activity in relation to human health and disease. Nevertheless there is a growing body of evidence identifying important roles of MMPs as regulators of myogenesis, fibrogenesis and adipogenesis. The aim of this review is to highlight the currently known functions of the MMPs that have a direct bearing on the deposition of meat components and their relationship with meat quality. Some central pathways by which these enzymes can affect the tenderness, the amount and type of fatty acids are highlighted.

Cytosolic Receptor Melanoma Differentiation-Associated Protein 5 Mediates Preconditioning-Induced Neuroprotection Against Cerebral Ischemic Injury.

BACKGROUND AND PURPOSE: Preconditioning with poly-l-lysine and carboxymethylcellulose (ICLC) provides robust neuroprotection from cerebral ischemia in a mouse stroke model. However, the receptor that mediates neuroprotection is unknown. As a synthetic double-stranded RNA, poly-ICLC may bind endosomal Toll-like receptor 3 or one of the cytosolic retinoic acid-inducible gene-I-like receptor family members, retinoic acid-inducible gene-I, or melanoma differentiation-associated protein 5. Activation of these receptors culminates in type I interferons (IFN-α/ß) induction-a response required for poly-ICLC-induced neuroprotection. In this study, we investigate the receptor required for poly-ICLC-induced neuroprotection. METHODS: Toll-like receptor 3, melanoma differentiation-associated protein 5-, and IFN-promoter stimulator 1-deficient mice were treated with poly-ICLC 24 hours before middle cerebral artery occlusion. Infarct volume was measured 24 hours after stroke to identify the receptor signaling pathways involved in protection. IFN-α/ß induction was measured in plasma samples collected 6 hours after poly-ICLC treatment. IFN-ß-deficient mice were used to test the requirement of IFN-ß for poly-ICLC-induced neuroprotection. Mice were treated with recombinant IFN-α-A to test the role of IFN-α as a potential mediator of neuroprotection. RESULTS: Poly-ICLC induction of both neuroprotection and systemic IFN-α/ß requires the cytosolic receptor melanoma differentiation-associated protein 5 and the adapter molecule IFN-promoter stimulator 1, whereas it is independent of Toll-like receptor 3. IFN-ß is not required for poly-ICLC-induced neuroprotection. IFN-α treatment protects against stroke. CONCLUSIONS: Poly-ICLC preconditioning is mediated by melanoma differentiation-associated protein 5 and its adaptor molecule IFN-promoter stimulator 1. This is the first evidence that a cytosolic receptor can mediate neuroprotection, providing a new target for the development of therapeutic agents to protect the brain from ischemic injury.

Changes in illness perceptions mediated the effect of cognitive behavioural therapy in severe functional somatic syndromes.

OBJECTIVE: Although there is substantial evidence that cognitive behavioural therapy alleviates symptoms in functional somatic syndromes, the mechanisms of change are less investigated. This study examined whether changes in illness perceptions mediated the effect of cognitive behavioural therapy. METHODS: We analysed additional data from a randomised controlled trial comparing completers of cognitive behavioural group therapy (46 patients) to an enhanced usual care group (66 patients). Proposed mediators (illness perceptions) and primary (physical health) and secondary (somatic symptoms and illness worry) outcomes were assessed by means of questionnaires at referral, baseline, end of treatment, and 10 and 16 months after randomisation. Multiple mediation analysis determined whether (1) changes in specific illness perceptions during treatment mediated the effect of cognitive behavioural therapy (primary analysis), and (2) whether changes in illness perceptions during the whole trial period were associated with improved outcome (secondary analysis). RESULTS: Improvements in illness perceptions during treatment partially mediated the effect of cognitive behavioural therapy on physical health one year after treatment (sum of indirect effects 1.556, BCa 95% CI (0.006; 3.620)). Improving perceived control was particularly important. Changes in illness perceptions from baseline to 16 months after randomisation were associated with clinically meaningful improvements in physical health, somatic symptoms and illness worry during the same period. CONCLUSION: Our results suggest that changing patients' illness perceptions is an important process in cognitive behavioural therapy for functional somatic syndromes. Challenging patients' own understanding of their illness may hence be a key element of successful treatment.

Percutaneous cholecystostomy is an effective treatment option for acute calculous cholecystitis: a 10-year experience.

BACKGROUND: Percutaneous cholecystostomy (PC) can be used to treat patients with acute calculous cholecystitis (ACC) who are considered to be unfit for surgery. However, this procedure has been insufficiently investigated. This paper presents the results of a 10-year experience with this treatment modality. METHODS: A retrospective observational study of all consecutive patients treated with PC for ACC in the period from 1 May 2002 to 30 April 2012 was conducted. All data were collected from patients' medical records. RESULTS: A total of 278 patients were treated with PC for ACC. Of these, 13 (4.7%) died within 30 days, 28 (10.1%) underwent early laparoscopic cholecystectomy and three (1.1%) patients were lost from follow-up. Of the remaining 234 patients, 55 (23.5%) were readmitted for the recurrence of cholecystitis. In 128 (54.7%) patients, PC was the definitive treatment (median follow-up time: 5 years), whereas 51 (21.8%) patients were treated with elective laparoscopic cholecystectomy. The frequency of recurrence of cholecystitis in patients with contrast passage to the duodenum on cholangiography was lower than that in patients without contrast passage (21.1% versus 36.7%; P = 0.037). CONCLUSIONS: The present study, which is the largest ever conducted in this treatment area, supports the hypothesis that PC is an effective treatment modality for critically ill patients with ACC unfit for surgery and results in a low rate of 30-day mortality.

Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1.

In many animals, including vertebrates, oocyte meiotic spindles are bipolar but assemble in the absence of centrosomes. Although meiotic spindle positioning in oocytes has been investigated extensively, much less is known about their assembly. In Caenorhabditis elegans, three genes previously shown to contribute to oocyte meiotic spindle assembly are the calponin homology domain protein encoded by aspm-1, the katanin family member mei-1, and the kinesin-12 family member klp-18. We isolated temperature-sensitive alleles of all three and investigated their requirements using live-cell imaging to reveal previously undocumented requirements for aspm-1 and mei-1. Our results indicate that bipolar but abnormal oocyte meiotic spindles assemble in aspm-1(-) embryos, whereas klp-18(-) and mei-1(-) mutants assemble monopolar and apolar spindles, respectively. Furthermore, two MEI-1 functions--ASPM-1 recruitment to the spindle and microtubule severing--both contribute to monopolar spindle assembly in klp-18(-) mutants. We conclude that microtubule severing and ASPM-1 both promote meiotic spindle pole assembly in C. elegans oocytes, whereas the kinesin 12 family member KLP-18 promotes spindle bipolarity.

Relative validity of micronutrient and fiber intake assessed with two new interactive meal- and Web-based food frequency questionnaires.

BACKGROUND: The meal- and Web-based food frequency questionnaires, Meal-Q and MiniMeal-Q, were developed for cost-efficient assessment of dietary intake in epidemiological studies. OBJECTIVE: The objective of this study was to evaluate the relative validity of micronutrient and fiber intake assessed with Meal-Q and MiniMeal-Q. The reproducibility of Meal-Q was also evaluated. METHODS: A total of 163 volunteer men and women aged between 20 and 63 years were recruited from Stockholm County, Sweden. Assessment of micronutrient and fiber intake with the 174-item Meal-Q was compared to a Web-based 7-day weighed food record (WFR). Two administered Meal-Q questionnaires were compared for reproducibility. The 126-item MiniMeal-Q, developed after the validation study, was evaluated in a simulated validation by using truncated Meal-Q data. RESULTS: The study population consisted of approximately 80% women (129/163) with a mean age of 33 years (SD 12) who were highly educated (130/163, 80% with >12 years of education) on average. Cross-classification of quartiles with the WFR placed 69% to 90% in the same/adjacent quartile for Meal-Q and 67% to 89% for MiniMeal-Q. Bland-Altman plots with the WFR and the questionnaires showed large variances and a trend of increasing underestimation with increasing intakes. Deattenuated and energy-adjusted Spearman rank correlations between the questionnaires and the WFR were in the range ρ=.25-.69, excluding sodium that was not statistically significant. Cross-classifications of quartiles of the 2 Meal-Q administrations placed 86% to 97% in the same/adjacent quartile. Intraclass correlation coefficients for energy-adjusted intakes were in the range of .50-.76. CONCLUSIONS: With the exception of sodium, this validation study demonstrates Meal-Q and MiniMeal-Q to be useful methods for ranking micronutrient and fiber intake in epidemiological studies with Web-based data collection.