CLC and IFNAR1 are differentially expressed and a global immunity score is distinct between early- and late-onset colorectal cancer.

Colorectal cancer (CRC) incidence increases with age, and early onset of the disease is an indication of genetic predisposition, estimated to cause up to 30% of all cases. To identify genes associated with early-onset CRC, we investigated gene expression levels within a series of young patients with CRCs who are not known to carry any hereditary syndromes (n=24; mean 43 years at diagnosis), and compared this with a series of CRCs from patients diagnosed at an older age (n=17; mean 79 years). Two individual genes were found to be differentially expressed between the two groups, with statistical significance; CLC was higher and IFNAR1 was less expressed in early-onset CRCs. Furthermore, genes located at chromosome band 19q13 were found to be enriched significantly among the genes with higher expression in the early-onset samples, including CLC. An elevated immune content within the early-onset group was observed from the differentially expressed genes. By application of outlier statistics, H3F3A was identified as a top candidate gene for a subset of the early-onset CRCs. In conclusion, CLC and IFNAR1 were identified to be overall differentially expressed between early- and late-onset CRC, and are important in the development of early-onset CRC.

The testicular germ cell tumour transcriptome.

Testicular germ cell tumours (TGCTs) are characterized by young age of onset and a complex pattern of histological subtypes. Transcriptomic studies have tried to uncover the gene expression patterns underlying this. Here, we present a systematic review of transcriptome studies of TGCTs of adolescents and young adults and identify genes common across the various studies, both for TGCTs in general as well as the histological subtypes, hence elucidating both transcriptional changes associated with malignant transformation and differentiation patterns. A meta-analysis of this type adds power and significance to the genes thus found, where most studies have included only a limited number of samples. Both known (KRAS, MYCN and TPD52) and novel (CCT6A, IGFBP3 and SALL2) cancer genes are implicated in TGC tumorigenesis. Gene expression patterns characteristic to embryonic stem cells are also found deregulated in TGC tumorigenesis. This is reflected in how pluripotent embryonal carcinoma cells commonly differentiate into a variety of embryonic and extra-embryonic histological types, each with unique transcriptomes. The embryonal carcinomas in particular are found to overexpress pluripotency genes, while gene signatures for seminomas, teratomas and yolk sac tumours were also identified. This underlines the distinctive transcriptomic programme across histological subtypes, especially striking given that the TGCT genome is largely similar across the same subtypes.

Drug-enhanced spinal stimulation for pain: a new strategy.

Neuropathic pain is notoriously difficult to manage and only a few classes of drugs may provide adequate benefits. Thus, in many cases spinal cord stimulation (SCS) is considered; however, in this group of patients, between 30-50% of the cases offered a percutaneous SCS trial may fail to obtain a satisfactory effect. Additionally, a certain number of patients with a good initial effect, report that after a period the benefits are reduced necessitating additional peroral drug therapy. Based on animal studies of transmitters and receptors involved in the effects of SCS in neuropathic pain, the GABA-B receptor seems to play a pivotal role for the effect and, moreover, the agonist baclofen injected intrathecally in rats potentiated the SCS effect in animals not responsive to SCS per se. Based on these and further studies, 48 patients with neuropathic pain and inadequate response to SCS were given intrathecal (i.t.) baclofen (ITB) in bolus doses as an adjuvant. In this group 7 patients enjoyed such a good effect that they were implanted with both SCS and drug delivery systems for ITB. Four additional cases received baclofen pumps alone. Some other patients were given intrathecal (i.t.) adenosine in combination with SCS and initially preferred this to baclofen. The chronic use of this drug in a pump however proved to be technically problematic and all the adenosine cases were eventually terminated. At follow-ups, in average 32 and 67 months after start of SCS + baclofen therapy, more than 50% still enjoy a very good effect. The daily dose of baclofen needed to maintain the effects was approximately doubled during the observation period. There were few and mild side-effects. However, in a group of three patients with peroral baclofen therapy and SCS, complaints of side-effects were common and this therapy was terminated. Informal reports from collegues support the negative experience with additional peroral baclofen. In conclusion, in patients with neuropathic pain demonstrating inadequate response to SCS (small VAS reduction; short duration) a trial of intrathecal baclofen in combination with SCS may be warranted.

Discrepancies between cortical and behavioural long-term readouts of hyperalgesia in awake freely moving rats.

BACKGROUND: It is still unclear to what extent the most common animal models of pain and analgesia, based on indirect measures such as nocifensive behaviours, provide valid measures of pain perception. METHODS: To address this issue, we developed a novel animal model comprising a more direct readout via chronically (>1 month) implanted multichannel electrodes (MCE) in rat primary somatosensory cortex (S1; known to be involved in pain perception in humans) and compared this readout to commonly used behavioural pain-related measures during development of hyperalgesia. A translational method to induce hyperalgesia, UVB irradiation of the skin, was used. Localized CO2 laser stimulation was made of twenty skin sites (20 stimulations/site/observation day) on the plantar hind paw, before and during the time period when enhanced pain perception is reported in humans after UVB irradiation. RESULTS: We demonstrate a 2-10 fold significant enhancement of cortical activity evoked from both irradiated and adjacent skin and a time course that corresponds to previously reported enhancement of pain magnitude during development of primary and secondary hyperalgesia in humans. In contrast, withdrawal reflexes were only significantly potentiated from the irradiated skin area and this potentiation was significantly delayed as compared to activity in S1. CONCLUSIONS: The present findings provide direct evidence that chronic recordings in S1 in awake animals can offer a powerful, and much sought for, translational model of the perception of pain magnitude during hyperalgesia. WHAT DOES THIS STUDY ADD?: In a novel animal model, chronic recordings of nociceptive activity in primary somatosensory cortex (S1) in awake freely moving rats are compared to behavioural readouts during UVB-induced hyperalgesia. Evoked activity in rat S1 replicates altered pain perception in humans during development of hyperalgesia, but withdrawal reflexes do not.

Frequent promoter hypermethylation of the O6-Methylguanine-DNA Methyltransferase (MGMT) gene in testicular cancer.

Testicular germ cell tumours are classified into two major histological subgroups, seminomas and nonseminomas. All tumours display several recurrent chromosomal aberrations, but few target genes have been identified. Previous studies have shown that genome-wide hypermethylation of CpG islands is significantly more prevalent in nonseminomas than in seminomas. We have studied two potential target genes in testicular cancer. A series of 70 tumours were analysed for methylation of CpG sites in the O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter, and in exon 1alpha of the cyclin-dependent kinase inhibitor 2A gene (CDKN2A). In addition, eight microsatellite markers within and flanking these genes at chromosome arms 10q and 9p, respectively, were analysed for allelic imbalances. Allele alterations were frequently seen at 9p loci (47 out of 70, 67%), but none of the tumours (none out of 55) showed methylation of CDKN2A. On the other hand, a high frequency of MGMT promoter methylation (32 out of 69, 46%) was found, as well as allelic imbalances at 10q markers (50 out of 70, 71%). A significantly higher methylation frequency was found in nonseminomas (24 out of 35, 69%) compared to seminomas (eight out of 33, 24%) (P=0.0003, Fisher's exact test). Immunohistochemical analysis of the MGMT protein in a subgroup (n=20) of the testicular tumours supported the hypothesis of gene silencing being the functional consequence of the promoter methylation. In summary, our data suggest that inactivation of MGMT contributes to development of nonseminomatous testicular cancer.

Nuclear muscarinic acetylcholine receptors in corneal cells from rabbit.

PURPOSE: Previous studies have indicated that muscarinic acetylcholine receptors (mAChR) may be present in an unexpected, unique location and play a singular role in cellular growth regulation of rabbit corneal epithelium that may be of general physiologic significance if found in other cells. The purpose of this study was to examine rabbit corneas and corneal cells in culture to determine mAChR location and tissue distribution. METHODS: Using [3H]-propylbenzilylcholine mustard ([3H]PrBChM), which binds covalently to the active site of mAChR, rabbit corneal cross-sections, cultured corneal keratocytes, epithelial and endothelial cells, as well as nuclei isolated from these cultured corneal cells were labeled, stained, and autoradiographed. Nuclei labeled with [3H]PrBChM were further analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Direct visual confirmation of the localization of mAChRs was obtained. MAChR were found in epithelial and endothelial layers of fresh-frozen corneal cross-sections, in cultured rabbit epithelial and endothelial cells, and on isolated rabbit epithelial and endothelial cell nuclei. mAChR were not detectable in keratocytes with these techniques. When [3H]PrBChM-labeled nuclei from cultured corneal cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, epithelial and endothelial samples showed specific mAChR binding, whereas binding to keratocyte nuclei was not detectable. CONCLUSIONS: As a result of these findings, a revised hypothesis is suggested for the locations and possible functions of mAChR in regulation of growth in corneal and other cells.

Identification and subcellular distribution of muscarinic acetylcholine receptor-related proteins in rabbit corneal and Chinese hamster ovary cells.

PURPOSE: The authors examined the muscarinic acetylcholine receptor (mAChR) subtypes in rabbit corneal epithelial and endothelial cells and in subcellular fractions of these cell types. A Chinese hamster ovary (CHO) cell line (nontransfected CHO K1), expected to be a negative control, also was investigated. METHODS: Whole cell homogenate and subcellular fractions were labeled with the covalent-binding, mAChR-specific ligand [3H]propylbenzilylcholine mustard ([3H]PrBChM) and were analyzed by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or SDS-PAGE, and autoradiography. RESULTS: A pattern of multiple PrBChM-binding proteins was detected in homogenates of corneal epithelial and endothelial cells and, surprisingly, in the CHO cells. Ligand binding to all of these proteins is inhibited by the mAChR antagonists atropine sulfate and quinculidinyl benzilate. The sizes of four of the labeled protein bands are the same as the molecular masses deduced from mAChR sequence data for subtypes m3, m4, m5, and either m1 or m2. One band of 47 kd, smaller than any reported sequence, was also observed. Two of the [3H]PrBChM-binding proteins, one at 59 to 62 kd (corresponding to m5 in size) and another at 47 kd, clearly were present when highly purified nuclei were analyzed. CONCLUSIONS: The presence of multiple mAChR-like proteins at low concentrations in these disparate cell types suggests the possibility of a more general regulatory role for this type of receptor than was considered previously. Combined with other reports, the identification of proteins with the characteristics of mAChRs in purified nuclei adds support to data indicating the likelihood of G-protein-coupled signaling across the nuclear envelope.

Muscarinic acetylcholine receptor antagonists inhibit chick scleral chondrocytes.

PURPOSE: Muscarinic acetylcholine receptors (mAChRs) have been implicated in the control of myopia in humans and in animal models. This study was conducted to determine whether mAChRs influence the growth of the chick sclera and, if so, which mAChR subtypes are involved. METHODS: Sclera and scleral chondrocytes from normal and form-deprived eyes of 10- to 14-day-old chicks were treated with a total of seven ligands: two agonists, carbachol (nonselective) and McN-A-343 (selective for the M1 mAChR subtype); and five antagonists, atropine (nonselective), pirenzepine and telenzepine (M1), gallamine (M2), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; M1 and M3). Incorporation of sulfate into glycosaminoglycans and of thymidine into DNA were quantified and normalized to sample DNA content. Possible toxicity of ligands at high doses was examined by analysis of cell number (by cell counting), viability (by trypan blue exclusion), and cellular metabolic activity (by dehydrogenase activity). RESULTS: Cellular proliferation and extracellular matrix production were inhibited by atropine in whole sclera and in its cartilaginous layer. Sulfate incorporation by chondrocytes from normal and form-deprived eyes was inhibited by mAChR antagonists with a rank order of potency (atropine > pirenzepine = 4-DAMP >> gallamine) consistent with regulation by M1, rather than M3 or M2 mAChR subtypes. Pirenzepine inhibited sulfate incorporation by chondrocytes from form-deprived eyes more effectively than those from normal eyes. Chondrocyte cultures were not viable when grown in high doses of any of the ligands used except gallamine. CONCLUSIONS: In chick scleral chondrocytes, synthesis of DNA and glycosaminoglycans was inhibited by mAChR antagonists. This inhibition was probably mediated by the M1 subtype mAChR. Therefore in vivo the sclera may be a site of action for the mAChR antagonists previously used to influence myopia. Although at high concentrations mAChR antagonists tested seemed to be toxic to chondrocytes, at lower doses inhibition occurred without toxic effects.

Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins.

PURPOSE: Recent studies of corneal wound healing suggest that activated corneal keratocytes develop myofibroblast-like characteristics including a putative contractile apparatus comprised, in part, of intracellular microfilament bundles (i.e., stress fibers) containing f-actin, myosin, and alpha-actinin; extracellular fibronectin fibrils; and fibronectin surface membrane receptors (alpha 5 beta 1 integrin). The purpose of this study was to determine the expression and organization of specific components of the contractile apparatus in normal, quiescent (in situ) corneal keratocytes, and to compare the in situ organization with that of activated, tissue culture (in vitro) corneal keratocytes that potentially mimic wound healing fibroblasts. METHODS: Cat corneal tissue was obtained immediately after sacrifice and was either fixed for in situ studies or cultured with MEM supplemented with 10% fetal calf serum for in vitro studies. Keratocytes (in situ and in vitro) were stained with the following probes: phalloidin, a mushroom toxin that specifically binds to f-actin; rabbit anti-bovine aortic myosin; monoclonal anti-human alpha-actinin; monoclonal anti-human vimentin; rabbit anti-human alpha 5 beta 1 integrin; monoclonal anti-human alpha 5 integrin; monoclonal anti-human connexin 43; and goat anti-human fibronectin. The cytoskeletal organization and co-localization were evaluated using epifluorescent and confocal microscopy. RESULTS: Normal, quiescent corneal keratocytes were distributed within the cornea as a lattice network, interconnected by broad, cellular processes extending from a flattened cell body. The f-actin distribution of in situ keratocytes was predominantly cortical and appeared to be closely associated with the plasma membrane. In addition, punctate areas that appeared to correlate with the localization of adhesion sites were identified. These punctate regions appeared to stain with antibodies to alpha 5 beta 1 but to not alpha 5. These data suggest that the fibronectin receptor, alpha 5 beta 1 integrin, is not present on normal corneal keratocytes. Based on co-localization studies, rabbit anti-bovine aortic myosin and monoclonal anti-alpha-actinin staining had similar distributions to FITC-phalloidin. Interconnections between keratocytes also showed staining for connexin 43, indicating the presence of gap junctions. By contrast, activated, cultured (in vitro) keratocytes showed an FITC-phalloidin staining pattern localized predominantly along intracellular stress fibers not detected in normal, quiescent keratocytes. Myosin and alpha-actinin staining had a similar stress fiber distribution, arranged in alternating bands and suggesting a sarcomeric distribution. Associated with stress fibers there was both anti-alpha 5 beta 1 and anti-alpha 5 staining, indicating the presence of focal adhesions. CONCLUSIONS: This study demonstrates that there are major structural differences in the organization of contractile cytoskeletal proteins between normal, quiescent (in situ), and activated (in vitro) keratocytes. In situ, contractile proteins appear to be associated with the cortical f-actin network, probably related to maintenance of cell shape and interconnectivity. Alternatively, activated keratocytes were characterized by the presence of a putative contractile apparatus comprised of f-actin, myosin, and alpha-actinin organized into sarcomeric, muscle-like bundles (stress fibers) associated with focal contacts containing alpha 5 beta 1 integrin. These data suggest that activation of keratocytes, i.e. myofibroblast transformation, must involve the reorganization of cytoplasmic contractile proteins as well as the expression of alpha 5 beta 1 integrin and the formation of focal contacts.

Role of G-proteins in ciliary process adenylyl cyclase responses of the albino rabbit eye.

After intravitreal injections of cholera or pertussis toxin (CTX or PTX, 0.5 -1 microgram/eye) into the albino rabbit eye, the in vitro responses of ciliary process adenylyl cyclase (AC) to isoproterenol, vasoactive intestinal peptide (VIP), and forskolin (FSK) were increased 21-40% for PTX, but for CTX-injected eyes AC responses to fluoroaluminate, VIP and FSK decreased 70-50%. The increased responses after PTX suggests that this toxin blocked an inhibitory Gi control of AC that is present in the control tissue. However, prolonged (> 24 hr) in vivo exposure to CTX appears to down-regulate the AC enzyme. In contrast to the in vivo findings, AC responsiveness was unaffected by PTX pre-treatment of membranes in vitro, while CTX pre-treatment increased basal activity (+600%), and the FSK response (+30%), but decreased responsiveness to fluoroaluminate, VIP and isoproterenol by 88-56%. Treatment of ciliary process membranes with 32P-NAD and CTX or PTX followed by SDS-PAGE autoradiography of labelled proteins gave two bands for the G-protein alpha-subunits of Gs (45, 56 kDa) and one broad band centered at 40 kDa for Gi-type subunits respectively. Western blots using specific antibodies showed the presence of Gi type I or III, but no detectable Gi type II or Go in rabbit ciliary processes. We conclude that the changes in adenylyl cyclase enzyme responses after intraocular CTX or PTX may not correlate with cAMP levels and intraocular pressure effects. However, the in vitro biochemical data on AC responses and on G-proteins provide evidence for dual regulation of ciliary process AC by activating and inhibitory G-proteins.

Biodegradation in laboratory activated sludge plants and aquatic toxicity of herbicides.

The biodegradation and the aquatic toxicity of four herbicides (isoproturon, terbuthylazine, mecoprop, metamitron) were investigated. Laboratory activated sludge plants were used for biodegradation experiments. The biodegradation of mecoprop reached nearly 100%, the other herbicides were not eliminated by biodegradation. The acute Daphnia magna 24-h assay, the algal 72-h inhibition test, and the recently developed lemna growth inhibition 7-d test were applied to evaluate the biological effects of herbicides as original substances. EC 50 and EC 10 values were determined. Algal and lemna test show that isoproturon and terbuthylazine are both much more toxic than mecoprop and metamitron. Daphnids are generally less sensitive against herbicides than plants. Biodegradation and toxicity test were coupled for mecoprop to assess biological long-term effects of possible biodegradation products of this herbicide. The effluents of the laboratory activated sludge units were used in toxicity tests (Daphnia magna 21-d reproduction test, lemna growth inhibition 7-d test). No inhibiting effect on the tested organisms was observed.

Biodegradation and aquatic toxicity of beta-alaninediacetic acid (beta-ADA).

The aquatic toxicity and biodegradability of the new chelating agent beta-alaninediacetic acid (beta-ADA) were investigated. There is no inhibition effect of beta-ADA in the daphnia magna 24 h test up to a concentration of 1000 mg/L. The algal growth inhibition test resulted in an EC 50 of 19.7 mg/L. An EC 20 of 740 mg/L was determined in the luminescent bacteria test. An EC 50 was not obtained in this test up to a concentration of 2000 mg/L beta-ADA. The degree of biodegradation of beta-ADA was determined in a static and a continuous test. The beta-ADA removal reached 98% at the end of the test after eight weeks in the continuous test which was carried out with laboratory activated sludge units simulating a waste water treatment plant. Further, biodegradation and toxicity tests were coupled, i.e. the effluents of the laboratory activated sludge units were applied in the toxicity tests. A higher toxicity of the effluents of the test units in comparison with the control unit was not observed.

[Spinal cord stimulation in chronic neuropathic pain]. / Ryggmärgsstimulering vid kronisk neuropatisk smärta.

Seventeen years' experience of spinal cord stimulation in the treatment of chronic pain has shown it to be effective only in the case of neuropathic pain--in particular, pain due to lesions in peripheral nerves or posterior roots. In such cases, pharmacological treatment is often unsuccessful, and transcutaneous electrical nerve stimulation is only useful in certain cases. In a retrospective study of 84 patients followed for up to 16 years, 56 patients were still using their stimulators and reported continued pain relief. The majority suffered from peripheral neuralgia due to trauma or surgery and 72 per cent in that group enjoyed satisfactory relief. Trial stimulation via a temporary extension lead for at least 4-5 days is a prerequisite of good long-term results. It is concluded that spinal cord stimulation is an indispensable tool for treating chronic neuropathic pain, and it merits to be used more frequently.

Epigenetic and genetic features of 24 colon cancer cell lines.

Cell lines are invaluable biomedical research tools, and recent literature has emphasized the importance of genotype authentication and characterization. In the present study, 24 out of 27 cell line identities were confirmed by short tandem repeat profiling. The molecular phenotypes of the 24 colon cancer cell lines were examined, and microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) were determined, using the Bethesda panel mononucleotide repeat loci and two epimarker panels, respectively. Furthermore, the BRAF, KRAS and PIK3CA oncogenes were analyzed for mutations in known hotspots, while the entire coding sequences of the PTEN and TP53 tumor suppressors were investigated. Nine cell lines showed MSI. Thirteen and nine cell lines were found to be CIMP positive, using the Issa panel and the Weisenberger et al. panel, respectively. The latter was found to be superior for CIMP classification of colon cancer cell lines. Seventeen cell lines harbored disrupting TP53 mutations. Altogether, 20/24 cell lines had the mitogen-activated protein kinase pathway activating mutually exclusive KRAS or BRAF mutations. PIK3CA and PTEN mutations leading to hyperactivation of the phosphoinositide 3-kinase/AKT pathway were observed in 13/24 cell lines. Interestingly, in four cell lines there were no mutations in neither BRAF, KRAS, PIK3CA nor in PTEN. In conclusion, this study presents molecular features of a large number of colon cancer cell lines to aid the selection of suitable in vitro models for descriptive and functional research.

SPG20, a novel biomarker for early detection of colorectal cancer, encodes a regulator of cytokinesis.

Colorectal cancer is a common disease with high mortality. Suitable biomarkers for detection of tumors at an early curable stage would significantly improve patient survival. Here, we show that the SPG20 (spastic paraplegia-20) promoter, encoding the multifunctional Spartin protein, is hypermethylated in 89% of colorectal carcinomas, 78% of adenomas and only 1% of normal mucosa samples. SPG20 methylation was also present in a pilot series of stool samples and corresponding tumors from colorectal cancer patients. SPG20 promoter hypermethylation resulted in loss of mRNA expression in various cancer types and subsequent depletion of Spartin. We further showed that Spartin downregulation in cancer cells resulted in cytokinesis arrest, which was reversed when SPG20 methylation was inhibited. The present study identifies SPG20 promoter hypermethylation as a biomarker suitable for non-invasive detection of colorectal cancer, and a possible mechanism for cytokinesis arrest in colorectal tumorigenesis.