A standardized method for measuring intercellular spaces in esophageal biopsies in patients with suspected gastroesophageal reflux disease (the intercellular space ratio).

OBJECTIVE: Dilated intercellular space (DIS) in esophageal biopsies is regarded as a possible early sign of mucosal injury in gastroesophageal reflux disease (GERD). This study presents a standardized approach of intercellular space measurement. MATERIAL AND METHODS: Distal and proximal esophageal biopsies were taken from 19 patients with suspected GERD, and examined with TEM. A grid containing 150 line-crossing points was applied upon each photomicrograph. The number of points falling on the intercellular space was divided by the total number of points of the grid, thereby creating a ratio called the intercellular space ratio (ISR). The ISR method was validated with regard to intra- and interobserver agreement, and was compared to a widely used method for measuring intercellular space diameter developed by Tobey et al. (Tobey NA, Carson JL, Alkiek RA, Orlando RC. Dilated intercellular spaces: a morphological feature of acid reflux-damaged human esophageal epithelium. Gastroenterology 1996;111(5):1200-1205). The ISR was also compared to other markers for GERD. Results. Pearson's correlation coefficients for intra- and interobserver agreement were 0.91 (p < 0.001) and 0.82 (p < 0.001), respectively. The Pearson's correlation coefficient between the ISR and the intercellular space diameter according to Tobey et al., measured in the same micrographs, was 0.32 (p < 0.001). The proximal ISR correlated significantly with the distal ISR (Spearman's rho = 0.57, p = 0.010), and with heartburn symptom score (Spearman's rho = 0.50, p = 0.028). CONCLUSIONS: The ISR showed a high intraobserver and interobserver agreement. It also displayed good external validity when compared to other markers for gastroesophageal reflux. A rather poor correlation was however found between the ISR and the intercellular space diameter measured as described by Tobey et al.

Metabolic changes in psoriatic skin under topical corticosteroid treatment.

BACKGROUND: MR spectroscopy of intact biopsies can provide a metabolic snapshot of the investigated tissue. The aim of the present study was to explore the metabolic pattern of uninvolved skin, psoriatic skin and corticosteroid treated psoriatic skin. METHODS: The three types of skin biopsy samples were excised from patients with psoriasis (N = 10). Lesions were evaluated clinically, and tissue biopsies were excised and analyzed by one-dimensional 1H MR spectroscopy. Relative levels were calculated for nine tissue metabolites. Subsequently, relative amounts of epidermis, dermis and subcutaneous tissue were scored by histopathological evaluation of HES stained sections. RESULTS: Seven out of 10 patients experienced at least 40% reduction in clinical score after corticosteroid treatment. Tissue biopsies from psoriatic skin contained lower levels of the metabolites myo-inositol and glucose, and higher levels of choline and taurine compared to uninvolved skin. In corticosteroid treated psoriatic skin, tissue levels of glucose, myo-inositol, GPC and glycine were increased, whereas choline was reduced, in patients with good therapeutic effect. These tissue levels are becoming more similar to metabolite levels in uninvolved skin. CONCLUSION: This MR method demonstrates that metabolism in psoriatic skin becomes similar to that of uninvolved skin after effective corticosteroid treatment. MR profiling of skin lesions reflect metabolic alterations related to pathogenesis and treatment effects.

Oral health problems are associated with malnutrition in hospitalised adult patients.

BACKGROUND & AIMS: Hospitalised patients are especially vulnerable to malnutrition, which is associated with an increased risk of complications, leading to longer hospital stays, increased healthcare costs, and with a potentially negative effect on the prognosis. Poor oral health may make food intake difficult and contribute to poor nutritional status. The aim of the present cross-sectional study was to assess the occurrence of poor oral health and malnutrition in adult hospitalised patients, and further to investigate associations between oral health problems and malnutrition. METHODS: The Patient-Generated Subjective Global Assessment (PG-SGA) determined the patients' nutritional status. The oral health condition was evaluated according to the Revised Oral Assessment Guide-Jönköping (ROAG-J) and unstimulated salivary flow rate. Clinical information was collected from medical records. RESULTS: The study population included 118 patients from 15 somatic and 3 psychiatric wards at a University Hospital in Norway. Nearly half the patients (46%) were categorised as malnourished and in need of symptom alleviation or nutritional intervention. Malnutrition was found in all diagnostic conditions. According to ROAG-J, at least one oral health problem was identified in 93% of the patients. Severe oral health problems were more frequent in malnourished patients. Overall, both the number and total score of oral health problems were associated with malnutrition (OR 1.57, 95% CI 1.20-2.06 and OR 1.47, 95% CI 1.17-1.83, respectively). Of specific oral health items, problems with lips and mucous membranes were significantly associated with malnutrition. One-fifth of all patients had hyposalivation, but this was not associated with malnutrition. CONCLUSIONS: Oral health problems and malnutrition are commonly seen in hospitalised adult patients. The association between the two calls for raised awareness of oral health issues in assessing patients' nutritional status. Further study is required to clarify whether oral health problems constitute a causal factor in malnutrition.

Efficacy and safety of transgastric closure in natural orifice transluminal endoscopic surgery using the OTSC system and T-bar sutures: a survival study in a porcine model.

BACKGROUND: In natural orifice transluminal endoscopic surgery (NOTES), procedures are performed with an endoscope passed through a natural orifice. One of the most important factors that will determine the future of transgastric NOTES is obtaining a reliable closure of the access site. The aim of this study was to determine the efficacy and safety of transgastric closure using the over-the-scope clip (OTSC) system or T-bar sutures. METHODS: We performed a survival study that included 15 pigs. A standardized transgastric approach to the peritoneal cavity and a peritoneoscopy were performed. The gastrotomy was closed using the OTSC system or T-bar sutures. The gastrotomy closure was tested for leaks with the methylene blue test. All animals were observed for 2 weeks before they were sacrificed and necropsy was performed. Histopathological examination of tissue samples retrieved from the access sites was performed. RESULTS: There were no perioperative complications. The methylene blue test did not demonstrate any leakage of fluid. Necropsy after 2 weeks confirmed completeness of gastric closure in all animals with full-thickness healing and no spillage of gastric contents into the peritoneal cavity. No differences between the OTSC system and T-bar sutures were observed. CONCLUSION: We observed no differences between the efficacy and safety of the OTSC system and those of T-bar sutures used in closing gastric incisions in NOTES. Both methods are safe and effective.

The population genomic legacy of the second plague pandemic.

Human populations have been shaped by catastrophes that may have left long-lasting signatures in their genomes. One notable example is the second plague pandemic that entered Europe in ca. 1,347 CE and repeatedly returned for over 300 years, with typical village and town mortality estimated at 10%-40%.1 It is assumed that this high mortality affected the gene pools of these populations. First, local population crashes reduced genetic diversity. Second, a change in frequency is expected for sequence variants that may have affected survival or susceptibility to the etiologic agent (Yersinia pestis).2 Third, mass mortality might alter the local gene pools through its impact on subsequent migration patterns. We explored these factors using the Norwegian city of Trondheim as a model, by sequencing 54 genomes spanning three time periods: (1) prior to the plague striking Trondheim in 1,349 CE, (2) the 17th-19th century, and (3) the present. We find that the pandemic period shaped the gene pool by reducing long distance immigration, in particular from the British Isles, and inducing a bottleneck that reduced genetic diversity. Although we also observe an excess of large FST values at multiple loci in the genome, these are shaped by reference biases introduced by mapping our relatively low genome coverage degraded DNA to the reference genome. This implies that attempts to detect selection using ancient DNA (aDNA) datasets that vary by read length and depth of sequencing coverage may be particularly challenging until methods have been developed to account for the impact of differential reference bias on test statistics.

A new method to provide a fresh frozen prostate slice suitable for gene expression study and MR spectroscopy.

BACKGROUND: Fresh frozen tissue from radical prostatectomy specimens is highly valuable material for research on gene expression and cellular metabolites. The purpose of this study was to develop a standardized method to provide a representative high quality research sample from radical prostatectomy specimens without interfering with the routine histopathological procedure. METHODS: A complete transversal slice is collected and snap-frozen before formalin fixation and routine processing of the remaining gland. The freezing preserves the original geometric shape, thus allowing subsampling of specific cell populations without thawing. RNA was extracted from 53 cylindrical subsamples (diameter 3 mm, thickness 2 mm) from 16 consecutive frozen slices. The histological pattern was evaluated by microscopy of a cryosection from sample before further analysis. RESULTS: Using this novel harvesting method close to 400 slices have been collected. Whenever tumor was present in both adjacent surrounding hematoxylin-eosin sections, we found cancer in 88% of the frozen slices. The extracted RNA showed very high quality with a mean RNA integrity number of 9.16 (SD 0.53). The MR spectra showed metabolic profiles containing several resonances, which deserve further evaluation as possible biomarkers for prostate cancer. After MR analysis the RNA was still highly intact with a mean RNA integrity number of 8.40 (SD 1.53), which makes it possible to correlate transcriptomic and metabolomic profiles of the extracted samples. CONCLUSION: We present a safe and standardized method for procurement of a high quality fresh frozen prostate slice, suitable for gene expression analysis and MR spectroscopy.

RNA-Integrity and 8-Isoprostane Levels Are Stable in Prostate Tissue Samples Upon Long-Term Storage at -80°C.

Sampling of prostate tissue (n = 97) was performed in conjunction with planned radical prostatectomies, in collaboration with Biobank1®. The tissue used in this study was collected during the period 2003-2016, quickly frozen, and kept at -80°C until assayed in 2018. RNA extraction was performed with two different protocols (miRNeasy and mirVana™), and RNA quality was determined by measuring the RNA Integrity Number (RIN). The level of isoprostanes is widely recognized as a specific indicator of lipid peroxidation both in vitro and in vivo. The level of 8-isoprostane was measured because it is the main oxidation product of arachidonic acid, the most abundant phospholipid fatty acid. The level of 8-isoprostane was measured using enzyme immunoassay. There was no statistically significant difference in yield between the samples isolated with the mirVana protocol compared to the miRNeasy protocol. Average RIN was 2.8 units higher with the mirVana extraction protocol compared to the miRNeasy protocol (p < 0.001). For miRNeasy extractions, RINs were 7.1 for prostatectomies in 2005-2007 and 6.2 for those in 2018 (p < 0.001). For mirVana extractions, the difference in RIN score between the two groups regarding years of collection was not statistically significant. There was no significant increase in the levels of 8-isoprostane between the 2005-2007 samples and the 2018. The conclusion is that there is no oxidation of phospholipids with increasing storage time up to 15 years.

Quantification of metabolites in breast cancer patients with different clinical prognosis using HR MAS MR spectroscopy.

Absolute quantitative measures of breast cancer tissue metabolites can increase our understanding of biological processes. Electronic REference To access In vivo Concentrations (ERETIC) was applied to high resolution magic angle spinning MR spectroscopy (HR MAS MRS) to quantify metabolites in intact breast cancer samples. The ERETIC signal was calibrated using solutions of creatine and TSP. The largest relative errors of the ERETIC method were 8.4%, compared to 4.4% for the HR MAS MRS method using TSP as a standard. The same MR experimental procedure was applied to intact tissue samples from breast cancer patients with clinically defined good (n = 13) and poor (n = 16) prognosis. All samples were examined by histopathology for relative content of different tissue types and proliferation index (MIB-1) after MR analysis. The resulting spectra were analyzed by quantification of tissue metabolites (ß-glucose, lactate, glycine, myo-inositol, taurine, glycerophosphocholine, phosphocholine, choline and creatine), by peak area ratios and by principal component analysis. We found a trend toward lower concentrations of glycine in patients with good prognosis (1.1 µmol/g) compared to patients with poor prognosis (1.9 µmol/g, p = 0.067). Tissue metabolite concentrations (except for ß-glucose) were also found to correlate to the fraction of tumor, connective, fat or glandular tissue by Pearson correlation analysis. Tissue concentrations of ß-glucose correlated to proliferation index (MIB-1) with a negative correlation factor (-0.45, p = 0.015), consistent with increased energy demand in proliferating tumor cells. By analyzing several metabolites simultaneously, either in ratios or by metabolic profiles analyzed by PCA, we found that tissue metabolites correlate to patients' prognoses and health status five years after surgery. This study shows that the diagnostic and prognostic potential in MR metabolite analysis of breast cancer tissue is greater when combining multiple metabolites (MR Metabolomics).

Principal component analysis for the comparison of metabolic profiles from human rectal cancer biopsies and colorectal xenografts using high-resolution magic angle spinning 1H magnetic resonance spectroscopy.

BACKGROUND: This study was conducted in order to elucidate metabolic differences between human rectal cancer biopsies and colorectal HT29, HCT116 and SW620 xenografts by using high-resolution magnetic angle spinning (MAS) magnetic resonance spectroscopy (MRS) and for determination of the most appropriate human rectal xenograft model for preclinical MR spectroscopy studies. A further aim was to investigate metabolic changes following irradiation of HT29 xenografts. METHODS: HR MAS MRS of tissue samples from xenografts and rectal biopsies were obtained with a Bruker Avance DRX600 spectrometer and analyzed using principal component analysis (PCA) and partial least square (PLS) regression analysis. RESULTS AND CONCLUSION: HR MAS MRS enabled assignment of 27 metabolites. Score plots from PCA of spin-echo and single-pulse spectra revealed separate clusters of the different xenografts and rectal biopsies, reflecting underlying differences in metabolite composition. The loading profile indicated that clustering was mainly based on differences in relative amounts of lipids, lactate and choline-containing compounds, with HT29 exhibiting the metabolic profile most similar to human rectal cancers tissue. Due to high necrotic fractions in the HT29 xenografts, radiation-induced changes were not detected when comparing spectra from untreated and irradiated HT29 xenografts. However, PLS calibration relating spectral data to the necrotic fraction revealed a significant correlation, indicating that necrotic fraction can be assessed from the MR spectra.

[Epithelial-to-mesenchymal transition and prostate cancer]. / Epitelial-mesenkymal transisjon og prostatakreft.

BACKGROUND: Prostate cancer is highly prevalent and is a frequent cause of death in men. As for most other cancers the prognosis is largely determined by the occurrence of metastases. Future treatment of prostate cancer should focus on inhibition of the cancer cells' ability to invade surrounding tissues--and to metastasise. In order to develop such therapies, it is important to unveil the mechanisms that lead to an invasive phenotype. Development of invasive tumours resemble processes involved in embryonic development, e.g. during formation of the mesoderm. The latter is characterised by a sequence of events whereby epithelial ectodermic cells acquire a migratory phenotype, which directly parallels the formation of invasive behaviour in carcinomas. MATERIAL AND METHODS: The present review is based on articles published in well-recognized journals of high international ranking. Some of the considerations also draw on the authors' personal experience in clinical work and basic research. RESULTS: Gastrulation is the process in which the three types of tissue stem cells move to different areas in the embryo (morphogenetic movement) and form the basis for various tissues and organs. This overview calls attention to the fact that events during carcinoma development, are strikingly similar to cellular changes during gastrulation. INTERPRETATION: A thorough understanding of gastrulation may provide a fruitful framework for new insight into cancer cell invasion and metastasis. This knowledge may in turn be exploited to develop drugs with anti-invasive properties which could revolutionise the treatment of carcinoma of the prostate and other sites.

Increased levels of serum miR-148a-3p are associated with prostate cancer.

Prostate cancer (PCa) is one of the most common types of cancer and the fifth leading cause of death among men worldwide. The tools for diagnosing PCa have limited value, and to improve correct diagnosis there is a need for markers that can contribute to a more precise diagnosis, which would lead to proper treatment of only those patients who need it. Micro RNA (miRNA) plays a key role in the development of cancer and is therefore a potential marker for PCa. Next-generation sequencing was used to discover differences in miRNA expression between serum samples from PCa patients and healthy controls, and the results were validated by quantitative real-time polymerase chain reaction. Detection of the miRNA of interest was attempted in prostate tissue by in situ hybridization. All samples were collected in collaboration with Biobank1® . By miRNA sequencing of serum samples, significant expression of some miRNAs in patients with PCa and healthy controls was detected. This study showed that miR-148a-3p is upregulated in men with PCa, and the miRNA is differentially expressed in PCa patients compared to healthy controls. The results also showed that miR-148a-3p is located in prostate tissue.

Anthropomorphic liver phantom with flow for multimodal image-guided liver therapy research and training.

PURPOSE: The objective of this study was to develop a multimodal, permanent liver phantom displaying functional vasculature and common pathologies, for teaching, training and equipment development in laparoscopic ultrasound and navigation. METHODS: Molten wax was injected simultaneously into the portal and hepatic veins of a human liver. Upon solidification of the wax, the surrounding liver tissue was dissolved, leaving a cast of the vessels. A connection was established between the two vascular trees by manually manipulating the wax. The cast was placed, along with different multimodal tumor models, in a liver shaped mold, which was subsequently filled with a polymer. After curing, the wax was melted and flushed out of the model, thereby establishing a system of interconnected channels, replicating the major vasculature of the original liver. Thus, a liquid can be circulated through the model in a way that closely mimics the natural blood flow. RESULTS: Both the tumor models, i.e., the metastatic tumors, hepatocellular carcinoma and benign cyst, and the vessels inside the liver model, were clearly visualized by all the three imaging modalities: CT, MR and ultrasound. Doppler ultrasound images of the vessels proved the blood flow functionality of the phantom. CONCLUSION: By a two-step casting procedure, we produced a multimodal liver phantom, with open vascular channels, and tumor models, that is the next best thing to practicing imaging and guidance procedures in animals or humans. The technique is in principle applicable to any organ of the body.

Urokinase plasminogen activator receptor (uPAR) expression is reduced by tyrosine kinase inhibitors.

Previously we reported that tyrosine kinase inhibitors (TKI) produced a reduction in uPA expression in prostatic cancer cells, and that TKI-treated cells were less invasive compared to untreated cells. Nevertheless, no change in cell migration was observed when TKI-treated cells were supplied with external uPA, thus indicating more complex mechanisms leading to decreased cell invasion. uPAR expression was measured with an enzyme-linked immunosorbent assay (ELISA) in PC-3 and DU-145 prostate carcinoma cells treated with the two TKI genistein and AG-1478. uPAR mRNA levels were measured with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). uPAR immunocytochemistry was used to examine the receptor distribution in cells grown on a reconstituted basal lamina. Immunocytochemistry showed an intense uPAR immunostaining in invading cells, particularly in the leading edge membrane. Treatment with genistein and AG-1478 led to a decreased expression of uPAR in DU-145, but not in PC-3. Furthermore, a reduction of uPAR mRNA was found in TKI-treated DU-145 cells, while PC-3 was not affected. Our results indicate a possible role of TKI as cancer suppressors by acting as a regulator of uPAR expression.

The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase.

Proteolytic enzymes, and especially urokinase plasminogen activator (uPA), play an important role in tumour invasion and metastasis. Previously we demonstrated that the production of urokinase plasminogen activator (uPA) was decreased by several tyrosine kinase inhibitors (TKI) in two prostatic carcinoma cell lines. The effect of the two TKI genistein and tyrphostin AG-1478 was investigated in the prostate carcinoma cell lines PC-3 and DU-145. A reconstituted basal lamina (Matrigel) was used as a migration barrier. The production of matrix metalloproteinases (MMP) was also measured. Roles of plasminogen and uPA were examined. Cell invasion was increased by plasminogen, but this enhanced cell migration was counteracted by TKI treatment. The increased cell invasion induced by plasminogen was decreased by at least 60% in both cell lines when alpha-2 anti-plasmin was added to the assay. Cells in the absence of plasminogen were not affected by TKI. External uPA failed to regenerate the decreased cell invasion caused by TKI. The production of MMP was inhibited by both TKI. Our results indicate a possible role of TKI as inhibitors of cancer cell invasion by inhibiting uPA and MMP production.

Tyrosine kinase inhibitors alter adhesivity of prostatic cancer cells to extracellular matrix components.

Tyrosine kinase inhibitors (TKIs) are thought to have potential as a new generation of anti-cancer drugs. Since invasiveness, the main characteristic of malignant behaviour, is believed to depend on altered cell-matrix interactions, we investigated the effect of two potent TKIs, genistein and tyrphostin AG-1478, on the interaction of prostate cancer cells with extracellular matrix components. PC-3 and DU-145 cells were treated with various concentrations of genistein and tyrphostin AG-1478. Adhesion to extracellular matrix was assayed using fluorescence-labelled cells seeded on collagen type I, collagen type IV, fibronectin, laminin and vitronectin. The expression levels of integrin beta1, alpha2, alpha3 and alpha5 subunits were measured using flow cytometry of cells labelled with monoclonal murine antibodies. Genistein treatment reduced the ability of both cell lines to adhere to the matrix proteins tested. This effect was more pronounced for PC-3 cells than for DU-145 cells. Genistein treatment decreased the expression of beta1 integrins by 40% in PC-3 cells and 22% in DU-145. AG-1478 treatment slightly reduced the ability of DU-145 cells to adhere, but did not decrease PC-3 cell adhesion. Nevertheless, expression levels were reduced for most integrins tested, except the expression of alpha-5, for which no significant effect was measured. Our results point to a possible role of TKIs as suppressors of prostate carcinoma cell adhesion to extracellular matrix components, by acting as inhibitors of integrin expression.

Transcription levels of invasion-related genes in prostate cancer cells are modified by inhibitors of tyrosine kinase.

Previous reports have shown that genistein and tyrphostin AG-1478, two tyrosine kinase inhibitors (TKIs), exert multiple cellular effects in prostate carcinoma cells, e.g. a reduction in the production of urokinase plasminogen activator (uPA) and its receptor uPAR, and a decrease in the cells' ability to invade an artificial basement membrane. Microarray technology was used to measure alterations in mRNA levels caused by TKI treatment in two prostatic carcinoma cell lines, PC-3 and DU-145. Genistein treatment led to a reduction of at least 50% in 78 genes in PC-3, while 82 were twofold upregulated. In DU-145, the same treatment resulted in a 50% decreased transcript level in 120 genes, and increased expression in 25 genes. Tyrphostin AG-1478 produced a 50% reduction in mRNA levels in 58 genes in DU-145, whereas no alterations were demonstrated using the tyrphostin in PC-3 cells. Among the effects of TKIs, a lowered uPA and uPAR transcription was demonstrated in genistein-treated cells, while a few metalloproteinases (MMPs) were affected. Transcription of various integrin subunits was also downregulated overall. Several alterations in gene transcription were demonstrated in PC-3 and DU-145 after TKI treatment. This knowledge could be of importance in the search for new therapeutic strategies in prostate cancer treatment, and the interplay between the various effects needs to be investigated further.

Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner.

Paraffin-embedded tissue is an important source of material for molecular pathology and genetic investigations. We used DNA isolated from microdissected formalin-fixed, paraffin-embedded gastric tumors for mutation analysis of a region of the human gene for uracil-DNA glycosylase (UNG), encoding the UNG catalytic domain, and detected apparent base substitutions which, after further investigation, proved to be polymerase chain reaction (PCR) artifacts. We demonstrate that low DNA template input in PCR can generate false mutations, mainly guanine to adenine transitions, in a sequence-dependent manner. One such mutation is identical to a mutation previously reported in the UNG gene in human glioma. This phenomenon was not caused by microheterogeneity in the sample material because the same artifact was seen after amplification of a homogenous, diluted plasmid. We did not observe genuine mutations in the UNG gene in 16 samples. Our results demonstrate that caution should be taken when interpreting data from PCR-based analysis of somatic mutations using low amounts of template DNA, and that methods used to enrich putative subpopulations of mutant molecules in a sample material could, in essence, be a further amplification of sequence-dependent PCR-generated artifacts.