PEDF Is Associated with the Termination of Chondrocyte Phenotype and Catabolism of Cartilage Tissue.

Objective. To investigate the expression and target genes of pigment epithelium-derived factor (PEDF) in cartilage and chondrocytes, respectively. Methods. We analyzed the expression pattern of PEDF in different human cartilaginous tissues including articular cartilage, osteophytic cartilage, and fetal epiphyseal and growth plate cartilage, by immunohistochemistry and quantitative real-time (qRT) PCR. Transcriptome analysis after stimulation of human articular chondrocytes with rhPEDF was performed by RNA sequencing (RNA-Seq) and confirmed by qRT-PCR. Results. Immunohistochemically, PEDF could be detected in transient cartilaginous tissue that is prone to undergo endochondral ossification, including epiphyseal cartilage, growth plate cartilage, and osteophytic cartilage. In contrast, PEDF was hardly detected in healthy articular cartilage and in the superficial zone of epiphyses, regions that are characterized by a permanent stable chondrocyte phenotype. RNA-Seq analysis and qRT-PCR demonstrated that rhPEDF significantly induced the expression of a number of matrix-degrading factors including SAA1, MMP1, MMP3, and MMP13. Simultaneously, a number of cartilage-specific genes including COL2A1, COL9A2, COMP, and LECT were among the most significantly downregulated genes. Conclusions. PEDF represents a marker for transient cartilage during all neonatal and postnatal developmental stages and promotes the termination of cartilage tissue by upregulation of matrix-degrading factors and downregulation of cartilage-specific genes. These data provide the basis for novel strategies to stabilize the phenotype of articular cartilage and prevent its degradation.

Inhibition of SHH pathway mechanisms by arsenic trioxide in pediatric medulloblastomas: a comprehensive literature review.

Recent innovations in the genomic understanding of medulloblastomas have provided new ways to explore this highly invasive malignant brain cancer arising from the cerebellum. Among the four different medulloblastoma subgroups described to date, the sonic hedgehog (SHH) genetic pathway is the pathway activated in the tumorigenesis of medulloblastoma. SHH-related medulloblastomas are usually of nodular/desmoplastic histology and frequently occur in children under the age of three, an age group highly susceptible to the acute and long-term effects of treatment. Several new drugs aimed at SHH modulation are currently under development. This review focuses on the role of arsenic trioxide, a drug well established in clinical practice and probably an under-explored agent in medulloblastoma management, in the SHH pathway.

Molecular differentiation between osteophytic and articular cartilage--clues for a transient and permanent chondrocyte phenotype.

OBJECTIVE: To identify the molecular differences between the transient and permanent chondrocyte phenotype in osteophytic and articular cartilage. METHODS: Total RNA was isolated from the cartilaginous layer of osteophytes and from intact articular cartilage from knee joints of 15 adult human donors and subjected to cDNA microarray analysis. The differential expression of relevant genes between these two cartilaginous tissues was additionally validated by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemistry. RESULTS: Among 47,000 screened transcripts, 600 transcripts were differentially expressed between osteophytic and articular chondrocytes. Osteophytic chondrocytes were characterized by increased expression of genes involved in the endochondral ossification process [bone gamma-carboxyglutamate protein/osteocalcin (BGLAP), bone morphogenetic protein-8B (BMP8B), collagen type I, alpha 2 (COL1A2), sclerostin (SOST), growth arrest and DNA damage-induced gene 45ß (GADD45ß), runt-related transcription factor 2 (RUNX2)], and genes encoding tissue remodeling enzymes [matrix metallopeptidase (MMP)9, 13, hyaluronan synthase 1 (HAS1)]. Articular chondrocytes expressed increased transcript levels of antagonists and inhibitors of the BMP- and Wnt-signaling pathways [Gremlin-1 (GREM1), frizzled-related protein (FRZB), WNT1 inducible signaling pathway protein-3 (WISP3)], as well as factors that inhibit terminal chondrocyte differentiation and endochondral bone formation [parathyroid hormone-like hormone (PTHLH), sex-determining region Y-box 9 (SOX9), stanniocalcin-2 (STC2), S100 calcium binding protein A1 (S100A1), S100 calcium binding protein B (S100B)]. Immunohistochemistry of tissue sections for GREM1 and BGLAP, the two most prominent differentially expressed genes, confirmed selective detection of GREM1 in articular chondrocytes and that of BGLAP in osteophytic chondrocytes and bone. CONCLUSIONS: Osteophytic and articular chondrocytes significantly differ in their gene expression pattern. In articular cartilage, a prominent expression of antagonists inhibiting the BMP- and Wnt-pathway may serve to lock and stabilize the permanent chondrocyte phenotype and thus prevent their terminal differentiation. In contrast, osteophytic chondrocytes express genes with roles in the endochondral ossification process, which may account for their transient phenotype.

Transplanted chondrocytes inhibit endochondral ossification within cartilage repair tissue.

The aim of this study was to investigate the effect of transplanted chondrocytes on endochondral bone formation in cartilage repair tissue. In the knee joint of miniature pigs, cartilage lesions were treated by microfracturing and were then either left empty, covered with a collagen membrane, or treated by matrix-associated autologous chondrocyte transplantation. In control lesions, the subchondral bone plate was left intact (partial-thickness lesion). The repair tissues were analyzed after 12 weeks by histological methods focusing on bone formation and vascularization. The effect of chondrocytes on angiogenesis was assessed by in vitro assays. The presence of antiangiogenic proteins in cartilage repair tissue, including thrombospondin-1 (TSP-1) and chondromodulin-I (ChM-I), was detected immunohistochemically and their expression in chondrocytes and bone marrow stromal cells was measured by quantitative RT-PCR. Significant outgrowths of subchondral bone and excessive endochondral ossification within the repair tissue were regularly observed in lesions with an exposed or microfractured subchondral bone plate. In contrast, such excessive bone formation was significantly inhibited by the additional transplantation of chondrocytes. Cartilaginous repair tissue that resisted ossification was strongly positive for the antiangiogenic proteins, TSP-1 and ChM-I, which were, however, not detectable in vascularized osseous outgrowths. Chondrocytes were identified to be the major source of TSP-1- and ChM-I expression and were shown to counteract the angiogenic activity of endothelial cells. These data suggest that the resistance of cartilaginous repair tissue against endochondral ossification following the transplantation of chondrocytes is associated with the presence of antiangiogenic proteins whose individual relevance has yet to be further explored.

Laparoscopic partial posterior fundoplication improves poor oesophageal contractility in patients with gastrooesophageal reflux disease.

OBJECTIVE: To investigate the effect of partial posterior fundoplication on oesophageal contractility in patients with gastrooesophageal reflux disease (GORD). DESIGN: Follow-up study with 6 months of survey. SETTING: University hospital, Austria. SUBJECTS: 24 consecutive patients with GORD and poor oesophageal contractility. INTERVENTIONS: Laparoscopic partial posterior fundoplication. Oesophageal contractility was assessed manometrically. MAIN OUTCOME MEASURES: Changes in measurements of mean contraction amplitudes in the distal oesophagus, the number of contractions with amplitudes of less than 30 mmHg, the number of interrupted and simultaneous contractions, and the total number of defective contractions. RESULTS: 16 of the patients (67%) complained of dysphagia preoperatively, and none postoperatively. The mean (SEM) amplitudes in the distal oesophagus improved significantly (level 442.4 mmHg (3.5) compared with 31.8 mmHg (3.3), p = 0.03, and level 5-45.7 mmHg (3.8) compared with 32.6 mmHg (3.7), p = 0.02), the number of contractions with amplitudes below 30 mmHg decreased (18.0% (5.7) compared with 38.3% (6.2), p = 0.02), as did the number of interrupted or defected contractions (11.5% (3.6) compared with 26.3% (5.5), p = 0.03, and 29.5% (6.5) compared with 66.6% (5.1), p < 0.0001 respectively). There was no significant effect on the number of simultaneous waves (p = 0.11). CONCLUSIONS: Partial posterior fundoplication improves poor oesophageal body motility. This results in improvement of preoperative dysphagia.

Hand-assisted laparoscopic splenectomy for isolated splenic metastasis from an ovarian carcinoma: a case report with review of the literature.

A 56-year-old woman was found to have a solitary metastasis to the spleen from ovarian cancer. A hand-assisted laparoscopic splenectomy was carried out. The operative technique and advantages of this technique are discussed. Laparoscopy with mini laparotomy allows for manual manipulation of the spleen with the ability to control bleeding, and facilitates mobilization and removal of the specimen. Postoperative recovery is rapid. Review of the literature indicates that secondary metastasis to the spleen is quite common.

Reflux-induced apoptosis of the esophageal mucosa is inhibited in Barrett's epithelium.

BACKGROUND: Apoptosis maintains cell homeostasis. Altered apoptosis is involved in carcinogenesis. It was our aim to investigate whether reflux esophagitis may alter apoptosis in the esophageal mucosa and whether antireflux surgery may restore normal apoptosis. METHODS: Apoptosis was studied preoperatively and postoperatively in esophageal biopsies of 39 patients with various grades of reflux esophagitis and in Barrett's mucosa using the TUNEL method. Biopsies were also taken from lesions of the squamous epithelium adjacent to the Barrett's mucosa. RESULTS: Apoptosis increased with the severity of esophagitis. Apoptosis was low in Barrett's epithelium. Squamous epithelium adjacent to Barrett's mucosa showed increased apoptosis. After surgery apoptosis decreased in squamous epithelium, and it remained low in Barrett's epithelium. CONCLUSIONS: Apoptosis in reflux esophagitis may be protective against increased proliferation. Low apoptosis following antireflux surgery indicates that surgery is effective to prevent reflux-induced cell proliferation. Inhibition of apoptosis in Barrett's may promote carcinogenesis. This may not change following surgery.

The surgical option for gastroesophageal reflux disease.

Gastroesophageal reflux disease is a common condition. Most patients can be managed with medications, but patients with refractory disease, particularly those with an incompetent lower esophageal sphincter, should be referred for surgery. The open Nissen fundoplication cures >90% of patients of their symptoms. The laparoscopic approach was first applied for patients with gastroesophageal reflux disease in 1991, and since then numerous reports evaluating the early experience with this technique have been published with results similar to the open procedure. Over the last 5 years, 595 laparoscopic antireflux procedures have been performed by us. There was 1 mortality due to an unrecognized duodenal perforation. Splenic injury did not occur compared to an incidence of up to 8.5% for the open procedure. A total of 9 patients required conversion to the open procedure for perforation, bleeding, or dissection difficulties. However, in the last 350 cases no conversions have been necessary. Most patients are now being discharged from hospital on the day after surgery with some patients being discharged on the same day as surgery. The overall reoperation rate, both for early postoperative morbidity and for late poor outcome, was 3.9% with follow-up ranging from 2 months to 5 years. The laparoscopic Nissen fundoplication achieves the same short-term outcome as the open procedure with significantly less postoperative morbidity and a shorter hospital stay.

Metabolism of dextromethorphan in human liver microsomes: a rapid HPCL assay to monitor cytochrome P450 2D6 activity.

A new HPLC assay was developed to study dextromethorphan O-demethylation to dextrorphan in vitro using human liver microsomes to investigate the activity of the polymorphic monooxygenase cytochrome P450 2D6 (CYP 2D6). The separation of dextromethorphan and its main metabolite dextrorphan was performed on a polymeric C18 reversed-phase column with UV-detection using levallorphan as an internal standard. Liver samples from ten subjects were screened for dextrorphan formation whereby three groups with different abilities to metabolize dextromethorphan could be found. Seven microsomal preparations from extensive metabolizers showed an average dextrorphan formation rate of 298 +/- 68 pmol/mg protein.min, one sample was classified to belong to an intermediate dextromethorphan metabolizer (79 pmol/mg protein.min), whereas two samples of poor metabolizers exhibited significantly lower rates of dextromethorphan metabolism with values of 11 and 27 pmol/mg protein.min, respectively. This assay permits not only a fast in vitro screening for cytochrome P450 2D6 monooxygenase activity but is also an excellent tool to determine potential drug-drug interactions with this important metabolizing enzyme.

The production of an amyloidogenic metabolite of the Alzheimer amyloid beta precursor protein (APP) in thyroid cells is stimulated by interleukin 1 beta, but inhibited by interferon gamma.

Thyroid epithelial cells have been shown to have a high APP expression and to produce large amounts of its metabolic derivatives, namely secreted APPs and a potentially amyloidogenic 41-kDa C-terminal fragment. It was the aim of the present study to analyze how APP production and metabolism were regulated in human thyroid cells. The effects of three cytokines, interferon gamma (IFN gamma), interleukin 1beta (IL-1 beta) and transforming growth factor (TGF) beta, were investigated. Cell extracts and supernatants were studied by immunoblotting using specific N- and C-terminal APP antibodies. Quantification was performed by densitometric scanning. We demonstrate that IFN gamma has a strong suppressive effect on the production and metabolism of APP. From a concentration of 30 U/ml upwards it reduces the cellular APP content, decreases the amounts of secreted APPs and inhibits the generation of the 41-kDa amyloidogenic APP fragment. In contrast, IL-1 beta has a stimulatory influence on the generation of the amyloidogenic 41-kDa APP metabolite, but does not affect the cellular holoprotein or APPs. TGFbeta has no significant effect on APP. Our results demonstrate that cytokines can regulate APP production and metabolism in thyroid cells. IFN gamma is the first naturally occurring agent described to inhibit the generation of amyloidogenic APP fragments. It may be of relevance in preventing amyloid deposition during inflammatory processes in the thyroid gland, but may exert a similar protective effect in other non-neuronal and neuronal tissues.

Prevention and management of related complication due to laparoscopic operation: pneumothorax, pneumomediastinum and subcutaneous emphysema.

Essential prerequisite of laparoscopic operation is establishing and maintaining pneumoperitoneum with a certain pressure. But under certain circumstances CO2-gas insufflating could induce pneumothorax, pneumomediastinum and subcutaneous emphysema during the operation. to probe the mechanism of the above complications. 2 typical cases were reported with attempt to probe the mechanism of the development of such applications. We believe that high intra-abdominal pressure during operation is the primary cause of the complications. The essentials of prevention and management of such complications were proposed.

Comparative anterograde amnestic and anticonvulsant effects of two types of NMDA receptor antagonists: MK-801 and HA-966.

The anterograde amnestic effects of non-competitive NMDA antagonists MK-801 and HA-966 on classic fear conditioning in goldfish (Carassius auratus) were examined in a series of experiments. Experiments 1 and 2 contrasted the anterograde amnestic effects of MK-801, (+)HA-966, and (-)HA-966. Experiment 3 examined the effects of MK-801 and (+)HA-966 on the expression of conditioned responses. Experiments 4 and 5 investigated whether the potency of MK-801, (+)HA-966 or (-)HA-966 in blocking NMDA-induced convulsions paralleled their potency in producing amnesia. The results showed that MK-801 was more potent than (+)HA-966 in producing anterograde amnesia and impairing expression, while (-)HA-966 did not produce anterograde amnesia. The anticonvulsant potency of MK-801, (+)HA-966, and (-)HA-966 paralleled their amnestic potency. These findings suggested that MK-801 and (+)HA-966 produced anterograde amnesia by their specific antagonism of the NMDA receptor complex.

NMDA receptor antagonist MK-801 blocks learning of conditioned stimulus-unconditioned stimulus contiguity but not fear of conditioned stimulus in goldfish (Carassius auratus L.).

The blockade of learning of Pavlovian fear conditioning by the N-methyl-D-aspartic acid (NMDA)-receptor antagonist MK-801 was examined in 166 goldfish. In previously untrained fish, MK-801 blocked learning of a light-off or a tone conditioned stimulus (CS) paired with an electrical shock unconditioned stimulus (US). Pretraining on the light-off CS did not affect the rate of learning of the tone CS but protected the tone learning from disruption by MK-801. Switching from the light-off to the tone CS changed the identity of the CS but not its temporal contiguity with the US. Pretraining consisting of pseudoconditioning of the light-off CS did not protect subsequent tone learning from blockade by MK-801. Thus, the NMDA receptor functions are necessary for learning related to the temporal contiguity of the CS and US but not to the identity of the CS as a cue to the occurrence of the fearful effects of the US.

Protocols for pharmacists' intervention in a 160 bed hospital.

The structure of many hospital pharmacy departments has become decentralized in order to provide pharmacists and other health care professionals with access to patient records. This change in departmental structure decreases the manager's ability to supervise the clinical activities of staff pharmacists. A policy for pharmacist intervention was developed by the pharmacy department. The policy identifies actions to be taken in specific situations, thus serving to standardize care.

Nervus terminalis innervation of the goldfish retina and behavioral visual sensitivity.

The possibility that axon terminals of the nervus terminalis in the goldfish retina regulate visual sensitivity was examined psychophysically. Fish were classically conditioned to respond in darkness to a diffuse red light conditioned stimulus. Bilateral ablation of the olfactory bulb and telencephalon had no significant effect on response threshold which was measured by a staircase method. Retinopetal nervus terminalis fibres thus appear to play no role in maintaining scotopic photosensitivity.

Spatial discrimination in goldfish following bilateral tectal ablation.

Goldfish were classically conditioned to discriminate between right and left or nasal and temporal presentations of a spot-of-light conditioned stimulus (CS). After the conditioning, the fish were administered bilateral optic tectum ablation followed by weekly sessions of conditioning trials to test for retention or relearning of the discrimination response. As their behavioral photosensitivity is greatly decreased, the ablates were dark-adapted prior to each session and trials were administered in darkness. Right x left discrimination was retained postoperatively but the nasal x temporal discrimination was blocked. Sham-operated controls discriminated between the nasal and temporal CS when dark-adapted and tested in darkness. Subsequent transection of the optic nerves obliterated response to the CS, indicating that tectum ablates detect and respond to the CS retinally and not extraretinally. We conclude that memory of visual spatial learning is mediated by non-tectal brain structures and that the ablate can discriminate between right- and left-eye input but sees the CS too diffusely to distinguish its location within the monocular field.

A computer program for selecting animals for control and experimental groups in biochemical studies.

The standard method for selecting experimental animals for control and experimental groups is by a randomization process in which each animal is placed into one of the groups without looking at any of its individual differences. An alternative, active matching process is proposed by which three to five animals are chosen to form 'experimental units' that are closely matched with respect to average weight and with respect to distribution of weights (standard distribution values). Several such units are then randomly assigned to each control or treatment group and, at the end of the experiment, the animals within each unit are pooled prior to analysis. The merits of this approach and a computer algorithm for making the selections are described.

Comparative neurotoxicity of tubulin-binding drugs: inhibition of goldfish optic nerve regeneration.

Intraperitoneal or intraocular (io) injection of tubulin-binding drugs in goldfish, Carassius auratus L., inhibited axonal regeneration or restoration of functional synapses in optic axons following optic nerve crush. One eye was used to detect effects on regeneration and the other was kept intact to detect effects on maintenance of established optic circuits. Regeneration was assessed by measuring the time to reappearance of a visually evoked branchial suppression response. Three drugs, vincristine, vinblastine, and podophyllotoxin, administered semiweekly by ip injection, each inhibited regeneration at doses that did not impair maintenance of response. Similar results were previously reported for ip colchicine. Vincristine was several times more potent than podophyllotoxin or colchicine and 25 times more potent than vinblastine. Picropodophyllotoxin, an isomer of podophyllotoxin which has low affinity for tubulin, did not inhibit regeneration. The io experiments showed that maintenance of vision was reversibly inhibited by a single injection of 0.05 micrograms/g of colchicine but unaffected by 0.01 microgram/g, and that administration of the lower dose immediately following optic nerve crush inhibited regeneration. Intraocular lumicolchicine, a colchicine photoisomer which has low affinity for tubulin, did not inhibit maintenance or regeneration. In contrast, an io dose of vincristine sufficient to inhibit visual recovery also blocked maintenance of vision. Thus regeneration and maintenance effects could not be dissociated for io vincristine suggesting its mechanism of action on retinal cells is different. A conditioning lesion was shown to decrease the time to reappearance of the visually evoked branchial response following optic nerve crush, which indicates that it is a sensitive index of the rate of axonal outgrowth to the optic tectum.

Systemic colchicine inhibits goldfish optic nerve regeneration.

Experiments were carried out to further investigate the regenerating goldfish optic nerve as a preparation for screening drugs or environmental toxins for adverse effects on neuronal circuit development. Regeneration was induced by unilateral retrobulbar optic nerve crush, and the opposite eye was kept intact. The time to recovery of vision was measured, as an index of regeneration and neurotoxicity, by an improved behavioral technique. The visual stimulus was changed to eliminate extraretinal photoresponding and to permit testing for vision with the right or left eye independently in a trial. Visual recovery occurred within 14 to 25 days. Colchicine, a potent inhibitor of microtubules and axonal transport, was administered semiweekly by ip injection, as in earlier experiments, to study the efficacy of the protocol. The drug resulted in an inhibition of regeneration at doses up to 0.2 micrograms/g body wt which did not impair responding with the control eye. Administration of beta-lumicolchicine, a photoisomer of colchicine that is a weak inhibitor of microtubules or axonal transport, up to 2.0 micrograms/g body wt, had no effect on regeneration or maintenance of visual responding. The results support the thesis that regenerating circuits are more sensitive indicators of neurotoxicity than are established circuits and confirm that the regenerating optic nerve can be used to screen molecules that may impair neuronal circuit development in vivo and to measure their relative potency.

Cholesterol synthesis and nerve regeneration.

In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [3H]mevalonolactone, exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20,25-diazacholesterol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of diazacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled desmosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [3H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro, diazacholesterol did not inhibit optic nerve regeneration in vivo, as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.