Association of Quadriceps Muscle Fat With Isometric Strength Measurements in Healthy Males Using Chemical Shift Encoding-Based Water-Fat Magnetic Resonance Imaging.

Magnetic resonance-based assessment of quadriceps muscle fat has been proposed as surrogate marker in sarcopenia, osteoarthritis, and neuromuscular disorders. We presently investigated the association of quadriceps muscle fat with isometric strength measurements in healthy males using chemical shift encoding-based water-fat magnetic resonance imaging. Intermuscular adipose tissue fraction and intramuscular proton density fat fraction correlated significantly (P < 0.05) with isometric strength (up to r = -0.83 and -0.87, respectively). Reproducibility of intermuscular adipose tissue fraction and intramuscular proton density fat fraction was 1.5% and 5.7%, respectively.

The need for T2 correction on MRS-based vertebral bone marrow fat quantification: implications for bone marrow fat fraction age dependence.

Vertebral bone marrow fat quantification using single-voxel MRS is confounded by overlapping water-fat peaks and the difference in T2 relaxation time between water and fat components. The purposes of the present study were: (i) to determine the proton density fat fraction (PDFF) of vertebral bone marrow using single-voxel multi-TE MRS, addressing these confounding effects; and (ii) to investigate the implications of these corrections with respect to the age dependence of the PDFF. Single-voxel MRS was performed in the L5 vertebral body of 86 subjects (54 women and 32 men). To reliably extract the water peak from the overlying fat peaks, the mean bone marrow fat spectrum was characterized based on the area of measurable fat peaks and an a priori knowledge of the chemical triglyceride structure. MRS measurements were performed at multiple TEs. The T2 -weighted fat fraction was calculated at each TE. In addition, a T2 correction was performed to obtain the PDFF and the T2 value of water (T2w ) was calculated. The implications of the T2 correction were investigated by studying the age dependence of the T2 -weighted fat fractions and the PDFF. Compared with the PDFF, all T2 -weighted fat fractions significantly overestimated the fat fraction. Compared with the age dependence of the PDFF, the age dependence of the T2 -weighted fat fraction showed an increased slope and intercept as TE increased for women and a strongly increased intercept as TE increased for men. For women, a negative association between the T2 value of bone marrow water and PDFF was found. Single-voxel MRS-based vertebral bone marrow fat quantification should be based on a multi-TE MRS measurement to minimize confounding effects on PDFF determination, and also to allow the simultaneous calculation of T2w , which might be considered as an additional parameter sensitive to the composition of the water compartment.

MR-detected changes in liver fat, abdominal fat, and vertebral bone marrow fat after a four-week calorie restriction in obese women.

PURPOSE: To determine changes in the bone marrow fat fraction (BMFF) in obesity after dietary intervention in comparison with changes in abdominal fat, liver fat, and serum lipids. MATERIALS AND METHODS: Twenty obese (BMI 34.92 ± 3.8 kg/m(2) ) women participated in a 4-week dietary intervention of 800 kcal/d plus additional vegetables. They underwent anthropometric and blood value measurements before and after the intervention. Abdominal 3T MRI was performed to measure changes in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) volume and single-voxel magnetic resonance spectroscopy (MRS) to measure fat content changes in the liver and L5 vertebral body. RESULTS: The greatest relative change after dietary intervention was found in the liver (-40.3%), followed by VAT volume (-15.1%), serum lipids (-12.6 to -14.5%), and SAT volume (-8.5%). There were no statistically significant changes in BMFF after dietary intervention (P = 0.39), but absolute changes in the BMFF were positively associated with SAT volume (r = 0.489) and negatively associated with nonadipose tissue volume (r = -0.493) before dietary intervention. CONCLUSION: Bone marrow behaves differently compared to SAT volume, VAT volume, liver fat, and serum lipids after a 4-week dietary intervention in obesity and BMFF changes depend on abdominal tissue volumes before intervention.

Immunohistochemical staining of Ki-67 using the monoclonal antibody Ki-s11 is a prognostic indicator for laryngeal squamous cell carcinoma.

BACKGROUND: Proliferative activity has been shown to be of prognostic significance for several malignancies. Ki-67, a cell cycle associated antigen, is regarded as a promising proliferation marker. Very few results on the proliferative activity of head and neck cancer and their potential prognostic value are available. MATERIALS AND METHODS: The proliferative activity of 104 squamous cell carcinomas of the larynx (SCCL) was analyzed retrospectively with the monoclonal antibody Ki-S11 which specifically detects the Ki-67 antigen. Median follow-up time was 47 months. RESULTS: There was a statistically significant correlation (p<0.05) between histopathological grading, N-status and proliferative activity. There was also a significant difference for the 5-year survival between low and highly proliferating tumours. The patient group with low proliferating laryngeal cancer had a statistically (p<0.05) longer absolute and recurrence-free 5-year-survival time than patients with a highly proliferating cancer. CONCLUSION: These results show that Ki-67 staining of SCCL with Ki-S11 is a helpful prognostic indicator for squamous cell carcinoma of the larynx with a potential clinical application.

LOXL4 is a selectively expressed candidate diagnostic antigen in head and neck cancer.

Selective up-regulation of the mRNA of LOXL4, a member of the LOX matrix amine oxidase family, significantly correlated with lymph node metastases and higher tumour stages in head and neck squamous cell carcinomas (HNSCC). To evaluate the diagnostic and prognostic value of the protein we produced an antibody specific for LOXL4 and assessed the expression in 317 human HNSCC specimens. The LOXL4 protein was detected in 92.7% of primary tumours, in 97.8% of lymph node metastases and in affected oral mucosa with high-grade dysplasia, but was absent in various non-neoplastic tissues of the head and neck. TNM categories and overall survival did not link to grades of immunoreactivity. Studies in cultured primary hypopharyngeal HTB-43 carcinoma cells detected perinuclear and cell surface expression of LOXL4, but no nuclear localisation. Therefore, its interactive SRCR-domains and catalytic activity combined with tumour cell specific expression and cell surface associated location indicate multiple functions in tumour cell adhesion and interactions with the extracellular matrix. Our data suggest that LOXL4 is useful both as tumour marker and target in the treatment of HNSCC.

Gene expression profiling of head and neck squamous cell carcinoma using cDNA microarrays.

The molecular mechanisms causing the development of squamous cell carcinomas in the head and neck region are mostly unknown. Identification of molecular changes which are involved in carcinogenesis might play a key role in improving the diagnosis, therapy and prognosis of patients with carcinomas in the head and neck. The purpose of the study was to identify transcriptional alterations of apoptosis associated genes between normal mucosa and tumor tissue. We measured the mRNA expression of 408 apoptosis associated genes by microarray-technique in normal upper aerodigestive tract mucosa (n=4), and in cancer tissue (n=8) of squamous cell carcinomas of the upper aerodigestive tract. RT-PCR was performed to confirm the microarray results. A hierarchial cluster analysis, based on 22 selected marker-genes showing a separation of the two tissue types supports the hypothesis of a specific expression pattern associated with tumor development. Additionally, we found 11 genes associated with anti-apoptotic processes to be upregulated while 12 genes associated with proapoptotic functions as well as 5 DNA-replication and chromosome cycle associated genes were found to be downregulated in the tumor tissue. Furthermore 6 of 8 genes which are known to be associated with lymphocyte activation were upregulated in tumor edge tissue. These results represent a first step for the diagnostic use of microarrays in squamous cell carcinomas of the head and neck region and might improve the understanding of the molecular mechanisms of carcinogenesis in the head and neck region.

MR-Based Assessment of Bone Marrow Fat in Osteoporosis, Diabetes, and Obesity.

Bone consists of the mineralized component (i.e., cortex and trabeculae) and the non-mineralized component (i.e., bone marrow). Most of the routine clinical bone imaging uses X-ray-based techniques and focuses on the mineralized component. However, bone marrow adiposity has been also shown to have a strong linkage with bone health. Specifically, multiple previous studies have demonstrated a negative association between bone marrow fat fraction (BMFF) and bone mineral density. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) are ideal imaging techniques for non-invasively investigating the properties of bone marrow fat. In the present work, we first review the most important MRI and MRS methods for assessing properties of bone marrow fat, including methodologies for measuring BMFF and bone marrow fatty acid composition parameters. Previous MRI and MRS studies measuring BMFF and fat unsaturation in the context of osteoporosis are then reviewed. Finally, previous studies investigating the relationship between bone marrow fat, other fat depots, and bone health in patients with obesity and type 2 diabetes are presented. In summary, MRI and MRS are powerful non-invasive techniques for measuring properties of bone marrow fat in osteoporosis, obesity, and type 2 diabetes and can assist in future studies investigating the pathophysiology of bone changes in the above clinical scenarios.

MR-based assessment of body fat distribution and characteristics.

The assessment of body fat distribution and characteristics using magnetic resonance (MR) methods has recently gained significant attention as it further extends our pathophysiological understanding of diseases including obesity, metabolic syndrome, or type 2 diabetes mellitus, and allows more detailed insights into treatment response and effects of lifestyle interventions. Therefore, the purpose of this study was to review the current literature on MR-based assessment of body fat distribution and characteristics. PubMed search was performed to identify relevant studies on the assessment of body fat distribution and characteristics using MR methods. T1-, T2-weighted MR Imaging (MRI), Magnetic Resonance Spectroscopy (MRS), and chemical shift-encoding based water-fat MRI have been successfully used for the assessment of body fat distribution and characteristics. The relationship of insulin resistance and serum lipids with abdominal adipose tissue (i.e. subcutaneous and visceral adipose tissue), liver, muscle, and bone marrow fat content have been extensively investigated and may help to understand the underlying pathophysiological mechanisms and the multifaceted obese phenotype. MR methods have also been used to monitor changes of body fat distribution and characteristics after interventions (e.g. diet or physical activity) and revealed distinct, adipose tissue-specific properties. Lastly, chemical shift-encoding based water-fat MRI can detect brown adipose tissue which is currently the focus of intense research as a potential treatment target for obesity. In conclusion, MR methods reliably allow the assessment of body fat distribution and characteristics. Irrespective of the promising findings based on these MR methods the clinical usefulness remains to be established.

Automatic segmentation of abdominal organs and adipose tissue compartments in water-fat MRI: Application to weight-loss in obesity.

PURPOSE: To develop a fully automatic algorithm for abdominal organs and adipose tissue compartments segmentation and to assess organ and adipose tissue volume changes in longitudinal water-fat magnetic resonance imaging (MRI) data. MATERIALS AND METHODS: Axial two-point Dixon images were acquired in 20 obese women (age range 24-65, BMI 34.9±3.8kg/m(2)) before and after a four-week calorie restriction. Abdominal organs, subcutaneous adipose tissue (SAT) compartments (abdominal, anterior, posterior), SAT regions along the feet-head direction and regional visceral adipose tissue (VAT) were assessed by a fully automatic algorithm using morphological operations and a multi-atlas-based segmentation method. RESULTS: The accuracy of organ segmentation represented by Dice coefficients ranged from 0.672±0.155 for the pancreas to 0.943±0.023 for the liver. Abdominal SAT changes were significantly greater in the posterior than the anterior SAT compartment (-11.4%±5.1% versus -9.5%±6.3%, p<0.001). The loss of VAT that was not located around any organ (-16.1%±8.9%) was significantly greater than the loss of VAT 5cm around liver, left and right kidney, spleen, and pancreas (p<0.05). CONCLUSION: The presented fully automatic algorithm showed good performance in abdominal adipose tissue and organ segmentation, and allowed the detection of SAT and VAT subcompartments changes during weight loss.

Microbial pattern recognition causes distinct functional micro-RNA signatures in primary human monocytes.

Micro-RNAs (miRNAs) are short, non-coding RNAs that regulate gene expression post transcriptionally. Several studies have demonstrated the relevance of miRNAs for a wide range of cellular mechanisms, however, the current knowledge on how miRNAs respond to relevant external stimuli, e.g. in disease scenarios is very limited. To generate a descriptive picture of the miRNA network associated to inflammatory responses, we quantified the levels of 330 miRNAs upon stimulation with a panel of pro-inflammatory components such as microbial pattern molecules (flagellin, diacylated lipopeptide lipopolysaccharide, muramyl dipeptide), infection with Listeria monocytogenes and TNF-α as pro-inflammatory control in primary human monocytes using real time PCR. As a result, we found distinct miRNA response clusters for each stimulus used. Additionally, we identified potential target genes of three selected miRNAs miR-129-5p, miR-146a and miR-378 which were part of PAMP-specific response clusters by transfecting THP1 monocytes with the corresponding pre- or anti-miRNAs and microfluidic PCR arrays. The miRNAs induced distinct transcriptomal signatures, e.g. overexpression of miRNA129-5p, which was selectively upregulated by the NOD2-elicitor MDP, led to an upregulation of DEFB1, IRAK1, FBXW7 and IKK γ (Nemo). Our findings on highly co-regulated clusters of miRNAs support the hypothesis that miRNAs act in functional groups. This study indicates that miRNAs play an important role in fine-tuning inflammatory mechanisms. Further investigation in the field of miRNA responses will help to understand their effects on gene expression and may close the regulatory gap between mRNA and protein expression in inflammatory diseases.

The level of secretory leukocyte protease inhibitor is decreased in metastatic head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas (HNSCC) represent the sixth largest group among all human malignancies. However, the exact molecular mechanisms inducing the genesis and the progression of metastasis in these tumors are poorly understood. The identification of molecular alterations involved in metastasis of HNSCC might influence the value of clinical diagnostics, impact therapy strategies and finally improve the prognosis of the patients. The purpose of this study was to identify clinically relevant alterations at the transcriptional and translational levels, when comparing metastatic (N+) and non-metastatic (N0) primary HNSCC. Three transcripts HERPUD1, SLPI and RAD51 were selected for further validation based on their association with carcinogenesis and metastasis. Quantitative real-time-PCR was performed to determine the mRNA expression levels. For subsequent confirmation of the results, immunohistochemistry was performed applying a monoclonal anti-SLPI antibody on 121 HNSCC tumor specimens (N0, n=40; N+, n=81). In metastatic primary cancer, SLPI mRNA showed 5.9-fold lower expression in comparison with non-metastatic primary cancer (p=0.0092). Immunohistochemical staining revealed a fold change of -1.79 between the N+ and the N0 group (p=0.0002). The results presented here clearly indicate the repression of SLPI, measurable on both, mRNA and protein levels in metastatic primary HNSCC as compared to non-metastatic HNSCC. Therefore, it can be assumed that SLPI might have a substantial protective effect on the metastasis process of HNSCC.

Prognostic relevance of the proliferation marker REPP86 for laryngeal cancer.

BACKGROUND: There is a clear correlation between proliferative activity and the biological behavior of cancer, which might have an impact on the patients' prognosis and consequences for the individual therapy concept. REPP86 (restrictedly expressed proliferation-associated protein 86) is a proliferation-associated protein expressed in S-, G(2)- and M-phases of the cell cycle, regarded as a promising proliferation marker and has not yet been examined in squamous cell carcinoma of the larynx (SCCL). MATERIALS AND METHODS: REPP86 was analyzed retrospectively in 104 SCCL using the monoclonal antibody Ki-S2. Proliferative activity was correlated with tumor stage, histopathological grading, patients' survival and the results we recently published on Ki-67 staining in SCCL. Median follow-up time was 47 months. RESULTS: A significant correlation (p<0.05) between histopathological grading, N-status and proliferation activity was observed. The patient group consisting of low proliferating laryngeal cancer showed a statistically longer absolute (p<0.05) and relapse-free (p=0.001) 5-year survival time than the group with a high proliferating tumor. Compared to the Ki-67 staining results, the REPP86 antibody better predicts the relapse-free 5-year-survival. CONCLUSION: Our results indicate that REPP86 staining of SCCL with Ki-S2 is a helpful prognostic indicator for SCCL and better predicts the relapse-free survival than Ki-67 staining in SCCL.

Molecular and immunological aspects of p53 and p53-autoantibodies in head and neck squamous cell carcinoma.

The tumour suppressor protein p53 (wild-type = wt-p53) is of major importance in the genetic integrity of the cell. Mutations of the p53-gene (mt-p53) are the most frequent genetic aberrations identified in different tumour entities. As analyzed in a wide variety of human malignomas, mt-p53 evokes a specific immune response. Yet, the possible occurrence of p53-autoantibodies in patients with head and neck squamous cell carcinomas (HNSCC) correlated to p53-mutations, p53 in sera and p53-overexpression in tissue has not been previously investigated. For the first time, the p53 status in 24 HNSCC patients was analyzed in the present study. The following parameters were investigated: analysis of mutation frequency of the p53-gene by direct sequencing of the exons 5-9, immunohistochemical detection of p53, measurement of the wt- and mt-p53-protein in sera by ELISA and p53-autoantibodies in sera by ELISA. Mutations of the p53-gene were detected in four (17%) patients. Overexpression of wt-p53 was detected by immunohistochemistry in 18 out of 24 (75%) tumours. In 8 (33%) patients the p53-protein was also detectable in sera, whereas in just one of these eight patients p53-autoantibodies were detectable simultaneously. Overall 6 out of 24 (25%) patients were found to be positive for serum p53-autoantibodies. Of these 6 cases, 5 could be assigned to tumours with immunohistochemically measurable wt-p53-overexpression. There was no correlation between p53-overexpression in tissue and p53-protein levels in sera or between p53-autoantibody levels in sera, nor in mutation frequency of the p53-gene and p53-overexpression in tissue. The results presented herein support the hypothesis that strong accumulation of p53 in the tissue is an important prerequisite for development of p53-autoantibodies. However, there must be further, yet unknown factors that influence the p53-autoantibody production because p53-autoantibodies were not identified in sera in each case of p53-accumulation in the tissue.

Genomic response of the rat brain to global ischemia and reperfusion.

To identify genes that are involved in ischemia response of the brain, we have evaluated changes of gene expression in rat cerebrum after 15 min complete global ischemia, followed by reperfusion for 1 h, 6 h or 24 h. The expression profiles of approximately 30,000 transcripts from three subjects in each group (including sham-operated controls) were monitored employing oligonucleotide microarrays. About 20,000 transcripts were detectable in rat brains. The levels of 576 transcripts (approximately 2.9%) were significantly altered in response to experimental ischemia. 419 transcripts were up- and 157 downregulated; 39 transcripts changed after 1 h reperfusion, 174 after 6 h and 462 after 24 h. Results from quantitative real-time reverse transcription PCR of 18 selected genes showed excellent agreement with the microarray data. There is surprisingly little overlap between gene regulation patterns at different reperfusion times (only seven genes displayed significant changes in transcript levels at all reperfusion times. Several genes that were previously unknown to be involved in ischemia-response have been identified. Analyses of gene ontology patterns and the most strongly regulated transcripts showed that the immediate response to an ischemia/reperfusion is mediated by the induction of specific transcription factors and stress genes. Delayed gene expression response is characterised by inflammation and immune-related genes. These results support the hypothesis that the brain's response to ischemia is an active, specific and coordinated process.