Thore Hillig

Higher skeletal muscle α2AMPK activation and lower energy charge and fat oxidation in men than in women during submaximal exercise

5′AMP‐activated protein kinase (AMPK) is an energy sensor activated by perturbed cellular energy status such as during muscle contraction. Activated AMPK is thought to regulate several key metabolic pathways. We used sex comparison to investigate whether AMPK signalling in skeletal muscle regulates fat oxidation during exercise. Moderately trained women and men completed 90 min bicycle exercise at 60%. Both AMPK Thr172 phosphorylation and α2AMPK activity were increased by exercise in men (∼200%, P < 0.001) but not significantly in women. The sex difference in muscle AMPK activation with exercise was accompanied by an increase in muscle free AMP (∼164%, P < 0.01), free AMP/ATP ratio (159%, P < 0.05), and creatine (∼42%, P < 0.001) in men but not in women (NS), suggesting that lack of AMPK activation in women was due to better maintenance of muscle cellular energy balance compared with men. During exercise, fat oxidation per kg lean body mass was higher in women than in men (P < 0.05). Regression analysis revealed that a higher proportion of type 1 muscle fibres (∼23%, P < 0.01) and a higher capillarization (∼23%, P < 0.05) in women than in men could partly explain the sex difference in α2AMPK activity (r=−0.54, P < 0.05) and fat oxidation (r= 0.64, P < 0.05) during exercise. On the other hand, fat oxidation appeared not to be regulated via AMPK. In conclusion, during prolonged submaximal exercise at 60%, higher fat oxidation in women cannot be explained by higher AMPK signalling but is accompanied by improved muscle cellular energy balance in women probably due to sex specific muscle morphology.

Cytochrome P450 2C9 plays an important role in the regulation of exercise‐induced skeletal muscle blood flow and oxygen uptake in humans

Previous studies show that exercise‐induced hyperaemia is unaffected by systemic inhibition of nitric oxide synthase (NOS) and it has been proposed that this may be due to compensation by other vasodilators. We studied the involvement of cytochrome P450 2C9 (CYP 2C9) in the regulation of skeletal muscle blood flow in humans and the interaction between CYP 2C9 and NOS. Seven males performed knee extensor exercise. Blood flow was measured by thermodilution and blood samples were drawn frequently from the femoral artery and vein at rest, during exercise and in recovery. The protocol was repeated three times on the same day. The first and the third protocols were controls, and in the second protocol either the CYP 2C9 inhibitor sulfaphenazole alone, or sulfaphenazole in combination with the NOS inhibitor Nω‐monomethyl‐l‐arginine (l‐NMMA) were infused. Compared with control there was no difference in blood flow at any time with sulfaphenazole infusion (P > 0.05) whereas with infusion of sulfaphenazole and l‐NMMA, blood flow during exercise was 16 ± 4 % lower than in control (9 min: 3.67 ± 0.31 vs. 4.29 ± 0.20 l min−1; P < 0.05). Oxygen uptake during exercise was 12 ± 3 % lower (9 min: 525 ± 46 vs. 594 ± 24 ml min−1; P < 0.05) with co‐infusion of sulfaphenazole and l‐NMMA, whereas oxygen uptake during sulfaphenazole infusion alone was not different from that of control (P > 0.05). The results demonstrate that CYP 2C9 plays an important role in the regulation of hyperaemia and oxygen uptake during exercise. Since inhibition of neither NOS nor CYP 2C9 alone affect skeletal muscle blood flow, an interaction between CYP 2C9 and NOS appears to exist so that a CYP‐dependent vasodilator mechanism takes over when NO production is compromised.

Nitric oxide production is a proximal signaling event controlling exercise-induced mRNA expression in human skeletal muscle

Previous studies have described the magnitude and time course by which several genes are regu‐lated within exercising skeletal muscle. These include interleukin‐6 (IL‐6), interleukin‐8 (IL‐8), heme oxygen‐ase‐1 (HO‐1), and heat shock protein‐72 (HSP72), which are involved in secondary signaling and preservation of intracellular environment. However, the primary signaling mechanisms coupling contraction to transcription are unknown. We hypothesized that exercise‐induced nitric oxide (NO) production is an important signaling event for IL‐6, IL‐8, HO‐1, and HSP72 expression in muscle. Twenty healthy males participated in the study. By realtime PCR, mRNA levels for 11 genes were determined in thigh muscle biopsies obtained 1) before and after 2 h knee extensor exercise without (control) and with con‐comitant NO synthase inhibition (nitro‐L‐arginine methyl ester, L‐NAME, 5 mgkg_1);or 2) before and after 2 h femoral artery infusion of the NO donor nitroglycerin (NTG, 1.5 μgkg_1min_1). L‐NAME caused marked reductions in exercise‐induced expression of 4 of 11 mRNAs including IL‐6, IL‐8, and HO‐1. IL‐6 protein release from the study leg to the circulation increased in the control but not in the L‐NAME trial. NTG infusion significantly augmented expression of the mRNAs attenuated by L‐NAME. These findings advance the novel concept that NO production contributes to regulation of gene expression in muscle during exercise. Subsequently, we sought evidence for involvement of AMP‐activated kinase or nuclear factor kappa B, but found none.—Steensberg, A., Keller, C., Hillig, T., Frosig, C., Wojtaszewski, J. F. P., Pedersen, B. K., Pilegaard, H., Sander, M. Nitric oxide production is a proximal signaling event controlling exercise‐induced mRNA expression in human skeletal muscle. FASEB J. 21, 2683–2694 (2007)

In vitro validation of an ultra-sensitive scanning fluorescence microscope for analysis of Circulating Tumor Cells

Analysis of circulating tumor cells (CTC) holds promise of providing liquid biopsies from patients with cancer. However, current methods include enrichment procedures. We present a method (CytoTrack®), where CTC from 7.5 mL of blood is stained, analyzed and counted by a scanning fluorescence microscope. The method was validated by breast cancer cells (MCF‐7) spiked in blood from healthy donors. The number of cells spiked in each blood sample was exactly determined by cell sorter and performed in three series of three samples spiked with 10, 33 or 100 cells in addition with three control samples for each series. The recovery rate of 10, 33 and 100 tumor cells in a blood sample was 55%, 70% and 78%, percent coefficient of variation (CV%) for samples was 59%, 32% and 18%, respectively. None of the control samples contained CTC. In conclusion, the method has been validated to highly sensitively detect breast cancer cells in spiking experiments and should be tested on blood samples from breast cancer patients. The method could benefit from automation that could reduce the CV%, and further optimization of the procedure to increase the recovery.

Assessing HER2 amplification by IHC, FISH, and real-time polymerase chain reaction analysis (real-time PCR) following LCM in formalin-fixed paraffin embedded tissue from 40 women with ovarian cancer.

We compare HER2 receptor amplification analysis by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real‐time polymerase chain reaction (real‐time PCR) DNA copy‐number assay following laser capture microdissection (LCM) in formalin‐fixed paraffin embedded tissue from 40 women with verified ovarian cancer. We speculate that LCM should result in a more accurate assessment of HER2 amplification in our real‐time PCR assay compared with IHC and FISH. HER2 overexpression measured by IHC, FISH, or real‐time PCR was found in 5.0%, 5.0%, and 22.5%, respectively. HER2 negative results measured by IHC, FISH, or real‐time PCR were found in 95%, 92.5%, and 60.0%, respectively. Analysis failed for IHC, FISH, or real‐time PCR in 0%, 2.5%, or 17.5% of cases. Concordance between IHC and FISH, IHC and real‐time PCR, or FISH and real‐time PCR were 89.7%, 72.7%, or 78.1%, respectively. Only few ovarian cancer patients were HER2 overexpressed measured by IHC or FISH and thus could be eligible for antibody‐based therapy with trastuzumab (Herceptin). Interestingly, we find an increased number of HER2 positive patients by real‐time PCR analysis on microdissected cancer cells, suggesting a number of HER2 positive patients not detected by current methods. Thus, the concept of quantitative measurement of HER2 on microdissected cancer cells should be explored further.

Decrease by 50% of plasma IgA tissue transglutaminase antibody concentrations within 2 months after start of gluten-free diet in children with celiac disease used as a confirming diagnostic test.

Abstract Background Histological examination of small bowel biopsies is normally the gold standard for the diagnosis of celiac disease (CD). The objective of this study was to investigate whether the rate of decreases in elevated plasma IgA tissue transglutaminase antibody (IgA-tTG) and/or IgG deamidated gliadin peptides antibody (IgG − DGP) concentrations could be used as a confirming test for CD in children on a gluten-free diet (GFD) when biopsy was omitted in the diagnostic process. Methods In this retrospective study we compared children (≤18 years old) with a CD-confirming biopsy (n = 16) to children without a biopsy (n = 18). After initiation of GFD the antibody half-life (the time (T½) when the antibody concentration is 50% decreased) was determined in all children. Results Children with a biopsy (IgA-tTG, T½ = 1.9 months; IgG − DGP, T½ = 2.2 months) and children without a biopsy (IgA-tTG, T½ = 1.6 months; IgG − DGP, T½ = 2.7 months) had comparable T½ (mean) results (p < 0.05) supporting that all children had the CD diagnosis. Conclusions When biopsy was omitted a rapid rate of decrease in CD antibody concentrations confirmed the CD diagnosis in children on GFD. The half-lives (T½) of IgA-tTG were less than 2 months in CD children.

Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

BACKGROUND A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. METHODS Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. RESULTS Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. CONCLUSIONS A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.

Mapping of HLA– DQ haplotypes in a group of Danish patients with celiac disease

Abstract Background: A cost-effective identification of HLA– DQ risk haplotypes using the single nucleotide polymorphism (SNP) technique has recently been applied in the diagnosis of celiac disease (CD) in four European populations. The objective of the study was to map risk HLA– DQ haplotypes in a group of Danish CD patients using the SNP technique. Methods: Cohort A: Among 65 patients with gastrointestinal symptoms we compared the HLA– DQ2 and HLA– DQ8 risk haplotypes obtained by the SNP technique (method 1) with results based on a sequence specific primer amplification technique (method 2) and a technique used in an assay from BioDiagene (method 3). Cohort B: 128 patients with histologically verified CD were tested for CD risk haplotypes (method 1). Patients with negative results were further tested for sub-haplotypes of HLA– DQ2 (methods 2 and 3). Results: Cohort A: The three applied methods provided the same HLA– DQ2 and HLA– DQ8 results among 61 patients. Four patients were negative for the HLA– DQ2 and HLA– DQ8 haplotypes (method 1) but were positive for the HLA– DQ2.5-trans and HLA– DQ2.2 haplotypes (methods 2 and 3). Cohort B: A total of 120 patients were positive for the HLA– DQ2.5-cis and HLA– DQ8 haplotypes (method 1). The remaining seven patients were positive for HLA– DQ2.5-trans or HLA– DQ2.2 haplotypes (methods 2 and 3). One patient was negative with all three HLA methods. Conclusions: The HLA– DQ risk haplotypes were detected in 93.8% of the CD patients using the SNP technique (method 1). The sensitivity increased to 99.2% by combining methods 1 – 3.

The Endocytic Collagen Receptor, uPARAP/Endo180, in Cancer Invasion and Tissue Remodeling

uPARAP/Endo180 is a constitutively recycling endocytosis receptor of 180 kDa. It is a type-1 membrane protein and includes an N-terminal Cys-rich domain followed by a fibronectin type-II domain, eight C-type lectin-like domains, a transmembrane segment, and a small cytoplasmic domain. The receptor binds and internalizes collagen, which is then directed to lysosomal degradation. The internalization efficiency increases when the collagen is in a gelatin-like state and, in line with this notion, the uptake of defined 1/2 and 3/4 collagen fragments is more efficient than the internalization of intact collagen. Thus, uPARAP/Endo180 most likely has a preferential role in the clearance of precleaved collagen, occurring after the initial attack of a collagenolytic MMP. Mesenchymal cell types such as fibroblasts, osteoblasts, some endothelial cells, and some macrophages express uPARAP/Endo180, with a strong expression in areas with dominant collagen turnover, such as developing bone. PyMT mice, which develop genetically induced, invasive mammary tumors, have reduced tumor growth and increased tumor collagen content when uPARAP/Endo180 is absent due to gene inactivation. Cancer cells have not been found to express uPARAP/Endo180 but some of the same cell types that express the receptor in healthy tissue show a strong increase in expression when they take part in the stroma that surrounds the cancer islets in some invasive cancers. Thus, in some situations involving invasive growth, collagen clearance by uPARAP/Endo180 is likely to take active part in the outgrowth and escape of cancer cells from a confined tissue compartment.

Retracing Circulating Tumour Cells for Biomarker Characterization after Enumeration

Background Retracing and biomarker characterization of individual circulating tumour cells (CTCs) may potentially contribute to personalized metastatic cancer therapy. This is relevant when a biopsy of the metastasis is complicated or impossible to acquire. Methods A novel disc format was used to map and retrace individual CTCs from breast-cancer patients and nucleated cells from healthy blood donors using the CytoTrack platform. For proof of the retracing concept, CTC HER2 characterization by immunofluorescence was tested. Results CTCs were detected and enumerated in three of four blood samples from breast-cancer patients and the locations of each individual CTCs were mapped on the discs. Nucleated cells were retraced on seven discs with 96.6%±8.5% recovery on five fields of view on each disc. Shifting of field of view for retracing was measured to 4-29 μm. In a blood sample from a HER2-positive breast-cancer patient, CTC enumeration and mapping was followed by HER2 characterization and retracing to demonstrate downstream immunofluorescence analysis of the CTC. Conclusion Mapping and retracing of CTCs enables downstream analysis of individual CTCs for existing and future cancer genotypic and phenotypic biomarkers. Future studies will uncover this potential of the novel retracing technology.