Non-pathological Tissue Research Articles

SASP: roadblock for tissue re-organization

Senescence has been shown to prevent and promote tumorigenesis. [1, 2] These results are not so paradoxical. To develop and maintain, organisms rely on cellular growth and cell division. Each of these processes is spatiotemporally finely tuned and both are tightly coordinated to ensure organism homeostasis throughout life. As organism aged, deregulations of these processes appear leading to hyperplastic or degenerating diseases, such as cancer and Alzheimer disease, respectively [3]. Interestingly, these two aged-related diseases have been linked to a cellular response that yet, uncouples cellular growth from cell division: senescence. Senescence is a natural cellular response that can be triggered by various stimuli, such as telomere shortening, oncogenic stresses or unrepaired DNA damages [4]. Senescent cells grow but do not divide so that they are enlarged and restricted in number. In addition, as they do not proliferate due to the irreversible cell cycle arrest, they do not differentiate. Thus senescence modifies tissue homeostasis by profoundly impacting tissue architecture both physically and biologically. Such disorganisation leads to alteration of cell contacts thereby re-wiring cellular communication. To communicate, cells use physical interactions and diffusible factors. In that context, it is interesting to observe that senescent cells often release factors such as cytokines or growth factors. This is known as senescence associated secretory phenotype (SASP) [5]. Recently Acosta et al have shown that the TGFβ pathway mediates paracrine senescence in SASP and that this pathway and the BMP pathway are upregulated in such senescent cells [6]. Interestingly, these two pathways are involved in tissue morphogenesis during organism development. It is therefore tempting to suggest that one of the outcomes of senescence is tissue re-organisation, achieved via cell communication, to reach new homeostasis upon cellular stress. As a matter of fact, studies of senescent cancer cells suggest so. First, senescence has been shown to act as an anti-cancer barrier, both physically and biologically in preneoplastic tissue [1]. Secondly, it has been shown to promote tumorigenesis by favouring the emergence of cancer stem-like cells (CSLCs) [7]. CSLCs are rare quiescent cells. They niche in heterogeneous tumors and have, in contrast to the bulk tumor cells but similarly to normal stem cells, the ability to self renew and to differentiate. Thus, if tissue has to be re-organised upon senescence to gain minimal homeostasis for functioning, new cells have to emerge and differentiate. This can be achieved by stimulation of CSLCs by SASP factors released from senescent cancer cells. Of note, it remains unclear why CSLCs, unlike normal stem cells, do not senesce. In relation to their role in tissue architecture, it has been described that CSLCs preferentially develop, within the tissue mass, under hypoxic conditions. Interestingly, hypoxia has been shown to inhibit mTOR, which converts quiescent cells into senescent cells [8]. If experimentally verified, hypoxia could reinforce the intrinsic resistance of CSLCs by maintaining their quiescent state, while inhibiting mTOR and geroconversion of CSLCs from quiescence to senescence. It therefore appears, at least in pathological cancer tissue, that senescence, and SASP in particular, could play a pivotal role in tissue re-organisation upon cellular stress. As a consequence, depending on the cancer stage, i.e. to which extend tissue has to be re-organised upon cancer invasion, senescence could be pro or anti tumorigenic. As to whether this role in tissue re-organisation also occurs in non-pathological tissue remains to be investigated. Tissue re-organisation by senescence implies cellular communication through SASP factors. Therefore it will be interesting to investigate if senescence is accompanied by secretion of SASP factors in unicellular organisms. If not, this will strongly argue for a role of SASP in maintaining tissue homeostasis, via tissue re-organisation, in multicellular organisms.

Augmenter of liver regeneration, a protective factor against ROS-induced oxidative damage in muscle tissue of mitochondrial myopathy affected patients

Mitochondria-related myopathies (MM) are a group of different diseases defined by a varying degree of dysfunctions of the mitochondrial respiratory chain which leads to reactive oxygen species (ROS) generation followed by oxidative stress and cellular damage. In mitochondrial myopathy muscle tissue an overexpression of antioxidant enzymes has been documented probably as an attempt to counteract the free radical generation.We previously documented, in human non-pathological muscle fibres, the expression of the augmenter of liver regeneration (ALR), a sulfhydryl oxidase enzyme, whose presence is related to the mitochondria; indeed it has been demonstrated that ALR mainly localizes in the mitochondrial inter-membrane space. Furthermore we reported, in different experimental models, in vivo and in vitro, the anti-apoptotic and anti-oxidative capacities of ALR, achieved by up-regulating Bcl-2 anti-apoptotic family factors and the anti-apoptotic/anti-oxidative secretory isoform of clusterin (sClu).With the present study we aimed to determine ALR, Bcl-2 protein, clusterin and ROS expression in muscle tissue biopsies from MM-affected patients. Non-pathological muscle tissue was used as control. Enzymatic, histochemical, immunohistochemical and immune electron microscopy techniques were performed.The data obtained revealed in MM-derived muscle tissue, compared to non-pathological tissue, the over-expression of ROS, ALR and Bcl-2 and the induction of the nuclear, pro-apoptotic, isoform of clusterin (nCLU).

PWE-167 Identification of Altered Keratin Level in Cancerized Colonic Fields using Isobaric Tags for Relative and Absolute Quantification (ITRAQ) for Protein Profiling

IntroductionA cancerized field is an area of abnormal tissue in the vicinity of a cancer that macroscopically appears normal, and which may be responsible for neoplastic recurrence. Evidence from keratin-8-deficient...

Identification of Unique Gene Expression Profile in Children with Regressive Autism Spectrum Disorder (ASD) and Ileocolitis

Gastrointestinal symptoms are common in children with autism spectrum disorder (ASD) and are often associated with mucosal inflammatory infiltrates of the small and large intestine. Although distinct histologic and immunohistochemical properties of this inflammatory infiltrate have been previously described in this ASDGI group, molecular characterization of these lesions has not been reported. In this study we utilize transcriptome profiling of gastrointestinal mucosal biopsy tissue from ASDGI children and three non-ASD control groups (Crohn's disease, ulcerative colitis, and histologically normal) in an effort to determine if there is a gene expression profile unique to the ASDGI group. Comparison of differentially expressed transcripts between the groups demonstrated that non-pathologic (normal) tissue segregated almost completely from inflamed tissue in all cases. Gene expression profiles in intestinal biopsy tissue from patients with Crohn's disease, ulcerative colitis, and ASDGI, while having significant overlap with each other, also showed distinctive features for each group. Taken together, these results demonstrate that ASDGI children have a gastrointestinal mucosal molecular profile that overlaps significantly with known inflammatory bowel disease (IBD), yet has distinctive features that further supports the presence of an ASD-associated IBD variant, or, alternatively, a prodromal phase of typical inflammatory bowel disease. Although we report qPCR confirmation of representative differentially expressed transcripts determined initially by microarray, these findings may be considered preliminary to the extent that they require further confirmation in a validation cohort.

Reduced tissue hardness of trabecular bone is associated with severe osteoarthritis

This study investigated whether changes in hardness of human trabecular bone are associated with osteoarthritis.Twenty femoral heads extracted from subjects without musculoskeletal diseases (subject age: 49–83 years) and twenty femoral heads extracted from osteoarthritic subjects (subject age: 42–85 years) were tested. Sixty indentations were performed along the main trabecular direction of each sample at a fixed relative distance. Two microstructures were found on the indenting locations: packs of parallel-lamellae (PL) and secondary osteons (SO). A 25gf load was applied for 15s and the Vickers Hardness (HV) was assessed.Trabecular tissue extracted from osteoarthritic subjects was found to be about 13% less hard compared to tissue extracted from non-pathologic subjects. However, tissue hardness was not significantly affected by gender or age. The SO was 10% less hard than the PL for both pathologic and non-pathologic tissues. A hardness of 34.1HV for PL and 30.8HV for SO was found for the non-pathologic tissue. For osteoarthritic tissue, the hardness was 30.2HV for PL and 27.1HV for SO. In the bone tissue extracted from osteoarthritic subjects the occurrence of indenting a SO (28%) was higher than that observed in the non-pathological tissue (15%).Osteoarthritis is associated with reduced tissue hardness and alterations in microstructure of the trabecular bone tissue. Gender does not significantly affect trabecular bone hardness either in non-pathological or osteoarthritic subjects. A similar conclusion can be drawn for age, although a larger donor sample size would be necessary to definitively exclude the existence of a slight effect.

Dependence of trabecular structure on bone quantity: A comparison between osteoarthritic and non-pathological bone

Background The mechanical characterization of trabecular bone is related to its structure. In order to describe the trabecular structure and to study the mechanical behavior of the trabecular tissue, several parameters are presented in the literature. Some studies suggest a possible dependence of the structure on bone volume fraction; this dependence could bias the validity of previous studies. The problem increases its complexity when pathological bone such as osteoarthritic tissue is studied, where the organization of the trabecular structure could be different if compared to the non-pathological tissue. The primary aim of this study was to evaluate the dependence between trabecular structure and bone volume fraction. The secondary aim was to compare osteoarthritic and non-pathological bone considering the correlation between structure and bone volume fraction. Methods Sixty trabecular bone specimens were extracted from femoral heads of two groups of 30 Caucasian donors; an osteoarthritic group and a non-pathological group. Several structural parameters, such as bone volume fraction, direct trabecular thickness, fabric tensor eigenvalues and their normalizations, were calculated from micro-CT analysis. A statistical analysis was carried out to identify the dependences between structural parameters and bone volume fraction. The comparison between osteoarthritic bone and non-pathological bone was also performed. Findings Only the normalized eigenvalues of the fabric tensor were not correlated to bone volume fraction (R < 0.5). The first and second normalized eigenvalues were significantly different between osteoarthritic bone and non-pathological bone (respectively P < 0.05 and P < 0.001). Interpretation In conclusion, orientation and anisotropy of the trabecular structure do not depend on bone volume fraction. Moreover, differences in the first and second normalized fabric tensor eigenvalues suggest in the osteoarthritic group a structure more oriented along the main trabecular direction.

Abstract P1-02-01: Vasohibin-1 in Human Breast Carcinoma: A Potential Negative Feedback Regulator of Angiogenesis

Abstract Background: Vasohibin-1 is a recently identified negative feedback regulator of angiogenesis induced by vascular endothelial growth factor (VEGF)-A. This is the first study to evaluate immunoreactivity and mRNA expression of Vasohibin-1 in human breast disorders. Material and Methods: We examined 251 breast specimens including invasive ductal carcinoma (IDC: 148), ductal carcinoma in situ (DCIS:62), fibroadenoma (FA:16), inflammatory lesion (6), fibrocystic change(9) and non-pathological breast tissue(7). We immunolocalized vasohibin-1 and compared its immunoreactivity to that of VEGF-A, VEGF receptor 2 (Flk-1), CD31 and Ki-67. The correlation of vasohibin-1 immunoreactibity with overall survival (OS), and disease free survival (DFS) of the breast carcinoma patients was also evaluated. In addition, we evaluated Ki-67 and CD31, and Ki-67 and vasohibin-1 double-immunostaining for further characterization of neovascularization. We also evaluated mRNA expression of Vasohibin-1 using a real-time quantitative RT-PCR with LightCycler system. Results: Vasohibin-1 was detected in endothelial cells of human breast and its immunodensity was significantly higher in IDC and inflammatory lesion than the other types (P&amp;lt;0.001). As for DCIS, there was a significant positive correlation between vasohibin-1 expression level and nuclear grade or Van Nuys classification of carcinoma cells (P&amp;lt;0.001). In addition, a significant positive correlation was detected between vasohibin-1 and VEGF-A, Flk-1 or Ki-67 (P&amp;lt;0.001). There was also positive associations between vasohibin-1 and OS (P=0.004) and DFS (P&amp;lt;0.001). Results of double-immunostaining demonstrated the ratio of Ki-67-positive cells among vasohibin-1-positive endothelial cells (46.5%) was significantly higher than those among CD31-positive cells (23.5%). In addition, there was a significant positive correlation between Vasohibin-1 mRNA level and Ki-67 or high nuclear grade(P&amp;lt;0.001). Discussion: This is the first study demonstrating the status of vasohibin-1 in human breast lesions, which indicates that vasohibin-1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer. Vasohibin-1 is supposed to be induced in the downstream of VEGF-A/Flk-1 signaling pathway and considered an appropriate biomarker for intratumoral neovascularization. In addition, these results clearly indicated that vasohibin-1 expression may become one of the prognostic markers for metastasis and prognosis, and also become one of the appropriate biomarker of the potential of subsequent stromal invasion of carcinoma cells. Figures available in online version. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-02-01.

Volume to density relation in adult human bone tissue

Uniformity of tissue mineralisation is a strongly debated issue, due to its relation with bone mechanical behaviour. Bone mineral density (BMD) is measured in the clinical practice and is applied in computational application to derive material proprieties of bone tissue. However, BMD cannot identify if the variation in bone density is related to a modification of tissue mineral density (TMD), a change in bone volume or a combination of the two. This study was aimed to investigate whether TMD can be assumed as a constant in adult human bone (trabecular and cortical). A total number of 115 cylindrical bone specimens were collected. An inter-site analysis (96 specimens, 2 donors) was performed on cortical and trabecular specimens extracted from different anatomical sites. An intra-site study (19 specimens, 19 donors) was performed on specimens extracted from femoral heads. Bone volume fraction (BV/TV) was computed by means of a micro-computed tomography. Furthermore, ash density ( ρ ash) was measured. TMD was computed as the ratio between ρ ash and BV/TV. It was found that the TMD of trabecular (1.24±0.16 g/cm 3) and cortical (1.19±0.06 g/cm 3) bone were not statistically different ( p=0.31). Furthermore, the linear regression between ρ ash and BV/TV was statistically significant ( r 2=0.99, p<0.001). Intra- and inter-site analyses demonstrated that the mineral distribution was independent of the extraction site. The present study suggests that TMD can be assumed reasonably constant in non-pathological adult bone tissue. Consequently, it is suggested that TMD can be managed as a constant in computational models, varying only BV in relation to clinical densitometric analysis.

Ectopic breast tissue at the anal verge—an unusual finding

Dear Editor: Traditional theory postulated that ectopic breast tissue could arise at any point along the embryonic milk lines, which extend bilaterally from the axilla to the medial aspect of the upper thigh. In normal circumstances, the majority of the milk lines regress with only a small proportion remaining in the mid-thorax for future breast development. However, a single gene with variable expression regulates natural suppression of the milk lines, and consequently, if some sections fail to regress, accessory breast tissue could develop at any point along their path. The more recent theory suggested by van der Putte (1991) proposed the normal existence of mammary-like anogenital glands and dismissed the traditional theory of ectopic breast tissue. However, this theory does not appear to have been universally accepted with some authors suggesting the presence of both mammary-like glands and ectopic breast tissue in the anogenital region. Here, we report a case of a 59-year-old lady who presented to the outpatient clinic with an anal swelling. It had been present for years but had become more prominent over the preceding 6-months, and she had become aware of it when sitting down. The lump was tender but not pruritic and did not bleed. She was systemically well with no other gastrointestinal abnormality. Her past medical history included a total abdominal hysterectomy for menorrhagia aged 33, hypothyroidism, hypertension, hypercholesterolaemia, carpal tunnel syndrome, and excision of an epidermal exclusion cyst from her back aged 55. Examination revealed a 1×1-cm cystic lesion just below the anal verge at the 5 o'clock position. This was suspected to be a simple skin cyst, and she proceeded to surgical excision. Gross examination of the specimen demonstrated a skin ellipse measuring 13×10 mm with underlying tissue to a depth of 5 mm, and on sectioning, a cyst was present which contained clear fluid. Microscopic examination revealed a well-defined, partially cystic lesion within the dermis. There was a proliferation of ductal structures lined by a dual cell layer. Focally, there were areas of apocrine change, and the large cyst identified macroscopically was lined by apocrine type columnar cells. Several papillary projections were seen to extend into the cyst lumen and some fibrous, focally hyalinised stroma surrounded the ductular structures. The overlying epidermis was essentially normal, and there was no evidence of malignancy. The findings were in keeping with ectopic breast tissue with cystic degeneration. Following a review of the literature, this would appear to be only the second reported case of nonpathological ectopic breast tissue arising in the perianal region. Chan et al. (2007) previously reported a case of ectopic breast tissue that presented as an anal polyp, and there have been five previous reported cases of adenomas detected in the perianal region. More recently, Charfi et al. (2009) described a polypoid lesion in the Competing interests None.

Hypoxia inducible factors in cancer stem cells

Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). Under hypoxia, HIFs regulate a variety of pro-angiogenic and pro-glycolysis pathways. In solid cancers, regions of hypoxia are commonly present throughout the tissue because of the chaotic vascular architecture and regions of necrosis. In these regions, the hypoxic state fluctuates in a spatial and temporal manner. Transient hypoxic cycling causes an increase in the activity of the HIF proteins above what is typical for non-pathologic tissue. The extent of hypoxia strongly correlates to poor patient survival, therapeutic resistance and an aggressive tumour phenotype, but the full contribution of hypoxia and the HIFs to tumour biology is an area of active investigation. Recent reports link resistance to conventional therapies and the metastatic potential to a stem-like tumour population, termed cancer stem cells (CSCs). We and others have shown that within brain tumours CSCs reside in two niches, a perivascular location and the surrounding necrotic tissue. Restricted oxygen conditions increase the CSC fraction and promote acquisition of a stem-like state. Cancer stem cells are critically dependant on the HIFs for survival, self-renewal, and tumour growth. These observations and those from normal stem cell biology provide a new mechanistic explanation for the contribution of hypoxia to malignancy. Further, the presence of hypoxia in tumours may present challenges for therapy because of the promotion of CSC phenotypes even upon successful killing of CSCs. The current experimental evidence suggests that CSCs are plastic cell states governed by microenvironmental conditions, such as hypoxia, that may be critical for the development of new therapies targeted to disrupt the microenvironment.

Cytochrome 3A and 2E1 in human liver tissue: Individual variations among normal Japanese subjects

Aims The metabolism of drugs, xenobiotic compounds, and other endogenous/exogenous substrates generally begins with their oxidation through cytochrome P450 (CYP). The results of recent pharmacogenetic analyses have demonstrated CYP's polymorphisms to be related to individual differences in metabolism, but only a limited number of CYP3A4 and CYP2E1 variant alleles influence enzymatic activities. Therefore, CYP gene expression profiling of both normal and pathological human livers should provide critical information for an evaluation of the biological significance of CYPs. Main methods In our present study, we first characterized the individual differences in CYP3A4 and CYP2E1 expression levels among Japanese normal or non-pathological liver tissue obtained from autopsy or surgery using immunohistochemistry and quantitative RT-PCR array of phase I metabolic enzymes with combined laser capture microscopy and qPCR analysis. Key findings Both CYP3A4 and CYP2E1 mRNA and proteins were predominantly detected in hepatocytes surrounding central veins in normal liver, but there were marked individual differences in both CYP3A4 and CYP2E1 mRNA and proteins among the 23 Japanese subjects examined. Individual differences in CYP3A and CYP2E1 subtypes were also detected in the livers obtained from monozygotic neonatal Japanese female twins with different survival periods. CYP3A and CYP2E1-positive cells were decreased in number in non-pathological hepatocytes of diseased livers compared to those in disease-free livers from autopsy. Significance The above results suggest that individual differences in CYP3A4 and CYP2E1 exist among normal human liver tissues and in non-pathological hepatocytes between diseased and normal liver, and these differences may be important in evaluating the pharmacodynamics of various substances.

Vasohibin‐1 in human breast carcinoma: A potential negative feedback regulator of angiogenesis

Vasohibin-1 is a recently identified negative feedback inhibitor or suppressor of angiogenesis induced by vascular endothelial growth factor (VEGF)-A. The status of vasohibin-1 in human breast carcinoma has not been examined. We examined 151 breast specimens including 98 cases of invasive ductal carcinoma (IDC), 12 of ductal carcinoma in situ (DCIS), 16 of fibroadenoma (FA), six of inflammatory lesion, nine of fibrocystic change and seven of non-pathological breast tissue. We immunolocalized vasohibin-1 and compared its immunoreactivity to that of VEGF-A, basic fibroblastic growth factor (bFGF), VEGF receptor 2 (Flk-1), CD31, CD34 and Ki-67/MIB-1. The correlation of vasohibin-1 immunoreactivity with overall survival (OS), and disease-free survival (DFS) of the patients with breast carcinoma was also evaluated. In addition, we evaluated Ki-67 and CD31, and Ki-67 and vasohibin-1 double-immunostaining for further characterization of neovascularization. Vasohibin-1 was detected in endothelial cells of human breast and its immunodensity was significantly higher in IDC and inflammatory lesions than the other types (P<0.001). In addition, a significant positive correlation was detected between vasohibin-1 and VEGF-A, bFGF or Flk-1 (P<0.001). There was also positive associations between vasohibin-1 and OS (P=0.004) and between vasohibin-1 and DFS (P<or=0.001) in carcinoma cases. Results of double-immunostaining demonstrated the ratio of Ki-67-positive cells among vasohibin-1-positive endothelial cells (46.5%) was significantly higher than those among CD31-positive cells (23.5%). This is the first study demonstrating the status of vasohibin-1 in human breast lesions, which indicates that vasohibin-1 is associated with neovascularization and may especially play important roles in the regulation of intratumoral angiogenesis in human breast cancer.

Hemodynamics of Cerebral Aneurysms

The initiation and progression of cerebral aneurysms are degenerative processes of the arterial wall driven by a complex interaction of biological and hemodynamic factors. Endothelial cells on the artery wall respond physiologically to blood-flow patterns. In normal conditions, these responses are associated with nonpathological tissue remodeling and adaptation. The combination of abnormal blood patterns and genetics predisposition could lead to the pathological formation of aneurysms. Here, we review recent progress on the basic mechanisms of aneurysm formation and evolution, with a focus on the role of hemodynamic patterns.

Absence of allelic imbalance involving EMSY, CAPN5, and PAK1 genes in papillary thyroid carcinoma

Papillary thyroid cancer (PTC) accounts for 80% of all thyroid malignancies, and genetic alterations associated to its etiology remain largely unknown. Chromosomal band 11q13 seems to be one of the most frequently amplified regions in human cancer, providing several candidate genes that need detailed characterization. The aim of our study was to investigate the existence of allelic imbalance at EMSY, CAPN5, and PAK1, as candidate genes within 11q13.5-q14 region using a single nucleotide polymorphism-based analysis. We selected a panel of 9 polymorphisms that were analyzed in 41 thyroid carcinoma samples, their contralateral non-pathological tissue and 178 controls from the general population. We did not detect allelic imbalance at these loci in our series. However, we observed a difference in the EMSY-haplotype distribution among PTC patients when compared to controls (odds ratio=2.00; p=0.02). We conclude that 11q13.5-q14 is not imbalanced in PTC, but there is evidence suggesting that EMSY might be of relevance in PTC etiology.

Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood.

Cytoplasmic granules from activated natural killer (NK) and cytotoxic T lymphocytes (CTL) contain a pore-forming protein, perforin, and several homologous serine proteinases called granzymes. Expression of these proteins correlates with the cytolytic potential of cytotoxic lymphocytes. Using a panel of MoAbs specific for human granzyme A and B, respectively, expression of these proteinases in non-pathological lymphoid tissue and peripheral blood lymphocyte (PBL) subpopulations was investigated. Using immunohistochemistry and double stainings, the phenotype of granzyme-expressing cells in lymphoid tissue was investigated. Granzyme-positive cells were detected in all lymphoid tissues tested. No large differences in the number and distribution between granzyme A- and granzyme B-positive cells were observed. The highest number of positive cells was located in the red pulp of the spleen. Significant numbers were detected in tonsil, lymph nodes, liver and thymus. Low numbers were present in the lamina propria of non-inflamed stomach, small intestine and colon. Phenotypic analysis and cell sorting showed that most of the granzyme-positive cells in lymphoid tissue and PBL consisted of CD3-CD16+CD56+ lymphocytes. Hardly any granzyme-positive CD3+CD8+ CTL were present in peripheral blood. The synthesis of granzyme A as well as B by both CD3+CD16+CD56+ and CD3+CD8+ cells in peripheral blood was increased upon IL-2 stimulation. These results indicate that in normal lymphoid tissue the predominant cytolytic cell population is formed by the NK cells, and activated CTL are rare.

DNA Methylation Biomarkers for Blood-Based Colorectal Cancer Screening

Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%). The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

EpCAM expression in normal, non-pathological tissues

Epithelial Cell Adhesion Molecule (EpCAM) is a transmembrane glycoprotein that is associated with various cancers. Most normal, non-pathological epithelial tissue is EpCAM positive with the exception of epidermal keratinocytes, gastric parietal cells, myoepithelial cells, thymic cortical epithelial, and hepatocytes. However, during early liver development EpCAM expression is also observed. In our studies, we have demonstrated that EpCAM is expressed in non-pathological human livers on hepatic progenitors in livers of all donor ages, from 16 weeks gestation fetal livers to adult. Hepatic progenitors of the liver consist of the stem cells and their descendants, the hepatoblasts, that give rise to the hepatocytic and biliary lineages. Both hepatic stem cells and most hepatoblasts express EpCAM, but only hepatoblasts are alpha-fetoprotein positive. The percentage of EpCAM positive progenitors in human livers varies with donor age and is about 2.5% in the adult and 12.1% in fetuses. In vivo, hepatic stem cells have been found associated with the canals of Hering. Xeno-transplantation experiments with EpCAM positive human liver cells have revealed their potential for proliferation and differentiation to mature liver parenchymal cells.

Splicing Factor Tra2-β1 Is Specifically Induced in Breast Cancer and Regulates Alternative Splicing of theCD44Gene

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.

Brain antioxidant levels in hamsters during hibernation, arousal and cenothermia

Warming from hibernation to cenothermia involves intense metabolic activity coincident with large fluxes in blood flow and is considered to be a period of oxidative stress during which utilization of endogenous antioxidants prevents pathology. Very slow flow brain microdialysis enabled temperature independent sampling of the brain striatal extracellular fluid (ECF) during hibernation, arousal and cenothermia in Syrian hamsters ( Mesocricetus auratus). Brain tissue and dialysates were analyzed to provide the first profile of changes in ECF levels of ascorbate (AA), glutathione (GSH) and urate during hibernation and the transition to cenothermia. Brain tissue content of AA and GSH was unchanged between hibernation and cenothermia; however, arousal was associated with substantial oxidation of AA from the brain ECF and plasma compartments. ECF GSH increased during arousal. Brain tissue urate content was decreased 50% during hibernation. ECF urate levels were unchanged in hibernation and cenothermia but transiently increased 100% during arousal. These experiments demonstrate that arousal from hibernation is a suitable experimental model for examination of the mechanisms by which non-pathological tissue integrity is maintained in the face of the generation of free radicals during increasing metabolism, temperature and cerebral reperfusion.

Large-scale disruption of microtubule pathways in morphologically normal human spastin muscle

To investigate the molecular pathways disrupted by dominant spastin mutations in apparently unaffected skeletal muscle from patients with motor neuron disease (SPG4). The authors studied muscle of three individuals from two unrelated families affected by spastic paraplegia caused by spastin mutations. The authors compared RNA expression profiles to 7 normal and 13 pathologic muscle U95A profiles (Duchenne dystrophy, acute quadriplegic myopathy, and spinal muscular atrophy). Data were validated with U133A arrays with seven different control specimens. mRNA and protein confirmations were done for a subset of genes. Both nonsense and missense mutations in the spastin gene disrupted microtubule pathways in nonpathologic tissue, including microtubule dynamics, stability, exocytosis, and endocytosis. Normal muscle can be used to uncover biochemical perturbation in motor neuron disease. Altered microtubule metabolism in SPG4-linked hereditary spastic paraplegia patients leads to pathology of the long descending tracks of motor neurons that likely have a stringent need for efficient microtubular transport. As many inherited neurologic conditions show a systemic biochemical defect with disease limited to neurons, our data have broader implications for biochemical pathway studies of many neurologic disorders.