Glycosaminoglycan-Mediated Loss of Cathepsin K Collagenolytic Activity in MPS I Contributes to Osteoclast and Growth Plate Abnormalities

Mucopolysaccharidoses are a group of lysosomal storage diseases characterized by the build-up of glycosaminoglycans (GAGs) and severe skeletal abnormalities. As GAGs can regulate the collagenolytic activity of the major osteoclastic protease cathepsin K, we investigated the presence and activity of cathepsin K and its co-localization with GAGs in mucopolysaccharidosis (MPS) type I bone. The most dramatic difference between MPS I and wild-type mice was an increase in the amount of cartilage in the growth plates in MPS I bones. Though the number of cathepsin K-expressing osteoclasts was increased in MPS I mice, these mice revealed a significant reduction in cathepsin K-mediated cartilage degradation. As excess heparan and dermatan sulfates inhibit type II collagen degradation by cathepsin K and the spatial overlap between cathepsin K and heparan sulfate strongly increased in MPS I mice, the build up of subepiphyseal cartilage is speculated to be a direct consequence of cathepsin K inhibition by MPS I-associated GAGs. Moreover, isolated MPS I and Ctsk^−/− osteoclasts displayed fewer actin rings and formed fewer resorption pits on dentine disks, as compared with wild-type cells. These results suggest that the accumulation of GAGs in murine MPS I bone has an inhibitory effect on cathepsin K activity, resulting in impaired osteoclast activity and decreased cartilage resorption, which may contribute to the bone pathology seen in MPS diseases.Mucopolysaccharidoses are a group of inherited lysosomal storage diseases. They result from the diminished activity of certain lysosomal enzymes responsible for glycosaminoglycan degradation. The subsequent accumulation of glycosaminoglycans (GAGs) in tissues triggers various pathological features including mental retardation, short stature, dysostosis multiplex, and corneal clouding.¹ The extent of these pathological features depends on the severity of the disease, which can range from mild to a severe form resulting in death in early childhood. Mucopolysaccharidosis type I (MPS I) is caused from an accumulation of heparan sulfate (HS) and dermatan sulfate (DS) due to the inactivity of the lysosomal enzyme α-iduronidase (Idua). Patients with MPS I display the typical characteristics of MPS diseases, including skeletal abnormalities such as dysostosis multiplex and spinal misalignment. Although much work is being done on enzyme replacement therapy to treat the disease,² the cellular and molecular mechanisms remain largely unknown.Cathepsin K, a lysosomal cysteine protease highly expressed by osteoclasts, is responsible for a significant part of total bone resorption.³ The lack of functioning enzyme results in a condition known as pycnodysostosis in humans.⁴ Osteoclasts are not able to degrade the organic matrix and so undegraded collagen accumulates in the resorption lacuna and in cellular lysosomes.⁵ Patients with pycnodysostosis typically have decreased bone resorption accompanied by enhanced bone density and are dwarfs.^6,7 Although the cathepsin K-deficient murine model does not display the build-up of collagen in osteoclast lysosomes and the dwarfism phenotype,⁵ it mimics the human disorder in most other ways.Recently we have shown that GAGs play an important role in the collagenolytic activity of cathepsin K.^8,9 Chondroitin sulfate (CS) was shown to form a complex with cathepsin K, which enabled the enzyme to fully degrade type I collagen.¹⁰ However other GAGs, like DS and HS, were shown to inhibit the collagenolytic activity of cathepsin K.¹¹ As both DS and HS are present in high concentrations in MPS I tissue, we investigated the effect these concentrations would have on cathepsin K activity. During endochondral ossification, cathepsin K degrades type II collagen (cartilage) below the growth plate area to provide room for new bone to be laid down by osteoblasts. This process is essential for the normal development of long bones.¹² As patients with MPS I have short stature with severe problems in bone growth and development, it is tempting to speculate that an impairment of cathepsin K activity may contribute to the disease phenotype. Therefore, we investigated the colocalization of HS and cathepsin K at the growth plate, the subepiphyseal cartilage structure, and the degradation of type II collagen in MPS I murine bone, as compared with wild-type bone. We also looked at the effect of GAGs in MPS I murine osteoclast activity. We hypothesize that high concentrations of HS and DS in MPS I may inhibit the activity of cathepsin K in osteoclasts and that this coupled with lysosomal dysfunction in osteoclasts contributes to MPS I bone pathology.     

Cytopathological and chemico‐physical analyses of smears of mucosa surrounding oral piercing

Oral Diseases (2010) 16 , 160–166 Objective: The aim of this comparative study was to analyze cytopathologically and chemico‐physically the mucosa surrounding oral piercing to correlate results with adverse tissue signs. Materials and methods: The tongue superficial mucosa of 15 young subjects (control group) and the superficial mucosa surrounding oral piercing of 15 young subjects (test group, TG) were smeared on slides, Papanicolaou stained and analyzed under the optical microscope. Some smears were prepared for (back‐scattered) scanning electron microscope (SEM) and X‐ray microanalysis to study piercing fragments. Results: Smears of TG displayed a variable extent of bacterial cytolysis of epithelial cells, fungi, hyperkeratosis, parakeratosis, granulocyte infiltration, calcium formations and bacterial flora; the four last statistically significant (P < 0.05). Foreign bodies surrounded by keratinocytes were detected under both light and SEM. X‐ray microanalyses highlighted piercing alloy aggression, ion release and an inverse gradient of ion concentration inside keratinocytes. Conclusions: The pathological findings in smears correlated with adverse effects of oral piercing. Ion release may be related to direct toxic effects and belated reactions because of metal sensitization. A strict regulation of piercing is warranted.     

Data-entropy analysis of renal transplantation data

INTRODUCTION: The terms entropy and robustness are currently used by biomedical investigators to predict the risk of change in a system. The former is the mathematical identification of uncertainty about a system, while the latter is the likelihood of system stability. We conducted an entropy-based analysis of our renal transplantation data set. MATERIALS AND METHODS: The input variables in our model included donors and recipients, past medical history, and other clinical data. The output variables were 6- month, 1-year, and 2- year patient and graft survivals. Data-entropy analysis was performed with Ontonix s.r.l. software (www.ontonix.com). RESULTS: The total input and output entropy was 13.14 and 1.54, respectively. The mean input and output robustness was 39.14% and 29.54%. The robustness amplification index was 0.75. The minimum entropy of the input variables was reported for a history of myocardial infarction (0.07), vascular disease (0.1), bladder residual (0.13), or urologic surgery (0.15). The minimum entropy of the output variables was 0.20 for 6-month patient survival; 0.22 for 1-year patient survival; 0.25 for 6-month graft survival; 0.27 for 1-year graft survival; 0.28 for 2-year patient survival; and 0.32 for 2-year graft survival. CONCLUSION: Data-entropy analysis demonstrated a high stability of our transplantation data set. Nevertheless, long-term outcomes, especially those of graft survival, were slightly more unpredictable.     

IDEAL PAIN RELIEF FOLLOWING LAPAROSCOPIC CHOLECYSTECTOMY

In a previous report the effectiveness of intraperitoneal bupivacaine in reducing pain following laparoscopic cholecystectomy was demonstrated. Other methods of pain relief are commonly used but none has been compared following laparoscopic cholecystectomy. In two further studies we have compared the analgesic effect of intraperitoneal bupivacaine against wound infiltration with bupivacaine, and against intraperitoneal bupivacaine with the addition of a non-steroidal anti-inflammatory drug (NSAID) in patients undergoing laparoscopic cholecystectomy. Two consecutive studies were performed. In the first, patients in group 1 were given 20 ml of 0.25% bupivacaine into the peritoneal cavity; patients in group 2 were given 20 ml of 0.25% bupivacaine injected into the trocar wounds. In the second study, patients in group 1 were given 20 ml of 0.25% bupivacaine into the peritoneal cavity; patients in group 2 were given 20 ml of 0.25% bupivacaine into the peritoneal cavity and a diclofenac suppository (100 mg) one hour before surgery. Postoperative pain was assessed with a visual analogue pain scale. There was no difference in pain scores in the two groups in either study. Intraperitoneal bupivacaine is as effective as wound infiltration. The addition of an NSAID makes no difference in the reduction of postoperative pain following laparoscopic cholecystectomy.     

Development of a new access device for transgastric surgery

Flexible endoscope-based endoluminal and transgastric surgery for cholecystectomy, appendectomy, bariatric, and antireflux procedures show promise as a less invasive form of surgery. Current endoscopes and instruments are inadequate to perform such complex surgeries for a variety of reasons: they are too flexible and are insufficient to provide robust grasping and anatomic retraction. The lack of support for a retroflexed endoscope in the peritoneal cavity makes it hard to reach remote structures and makes vigorous retraction of tissues and organs difficult. There is also a need for multiple channels in scopes to allow use of several instruments and to provide traction/countertraction. Finally, secure means of tissue approximation are critical. The aim was to develop and test a new articulating flexible endoscopic system for endoluminal and transgastric endosurgery. A multidisciplinary group of gastrointestinal physicians and surgeons worked with medical device engineers to develop new devices and instruments. Needs assessments and design parameters were developed by consensus. Prototype devices were tested using inanimate models until usable devices were arrived at. The devices were tested in nonsurvival pigs and dogs. The devices were accessed through an incision in the wall of the stomach and manipulated in the peritoneal cavity to accomplish four different tasks: right upper quadrant wedge liver biopsy, right lower quadrant cecal retraction, left lower quadrant running small bowel, and left lower quadrant exposure of esophageal hiatus. In another three pigs, transgastric cholecystectomy was attempted. The positions of the device, camera, and endosurgical instruments, with and without ShapeLock technology, were recorded using laparoscopy and endoscopy and procedure times and success rates were measured. Instrument design parameters and their engineering solutions are described. Flexible multilumen guides which could be locked in position, including a prototype which allowed triangulation, were constructed. Features of the 18-mm devices include multidirectional mid body and/or tip angulation, two 5.5-mm accessory channels allowing the use of large (5-mm) flexible endosurgical instruments, as well as a 4-mm channel for an ultraslim prototype video endoscope (Pentax 4 mm). Using the resulting devices, the four designated transgastric procedures were performed in anesthetized animals. One hundred percent of the transgastric endosurgical procedures were accomplished with the exception of a 50% success for hiatal exposure, a 90% success rate for wedge liver biopsy, and a 33.3% success rate for cholecystectomy. A new endosurgical multilumen device and advanced instrumentation allowed effective transgastric exploration and procedures in the abdominal cavity including retraction of the liver and stomach to allow exposure of the gallbladder, retraction of the cecum, manipulation of the small bowel, and exposure of the esophageal hiatus. This technology may serve as the needed platform for transgastric cholecystectomy, gastric reduction, fundoplication, hiatus hernia repair, or other advanced endosurgical procedures. Presented at the Forty-Sixth Annual Meeting of The Society for Surgery of the Alimentary Tract, Chicago, Illinois, May 14–18, 2005 (oral presentation). Partially funded and supported by USGI Medical, San Clemente, California.     

Coagulation factor activity and clinical bleeding severity in rare bleeding disorders: results from the European Network of Rare Bleeding Disorders

Summary. Background: The European Network of Rare Bleeding Disorders (EN‐RBD) was established to bridge the gap between knowledge and practise in the care of patients with RBDs. Objectives: To explore the relationship between coagulation factor activity level and bleeding severity in patients with RBDs. Patients/Methods: Cross‐sectional study using data from 489 patients registered in the EN‐RBD. Coagulation factor activity levels were retrieved. Clinical bleeding episodes were classified into four categories according to severity. Results: The mean age of patients at data collection was 31 years (range, 7 months to 95 years), with an equal sex distribution. On linear regression analysis, there was a strong association between coagulation factor activity level and clinical bleeding severity for fibrinogen, factor (F) X, FXIII, and combined FV and FVIII deficiencies. A weaker association was present for FV and FVII deficiencies. There was no association between coagulation factor activity level and clinical bleeding severity for FXI. The coagulation factor activity levels that were necessary for patients to remain asymptomatic were: fibrinogen, > 100 mg dL^?1; FV, 12 U dL^?1; combined FV + VIII, 43 U dL^?1; FVII, 25 U dL^?1; FX, 56 U dL^?1; FXI, 26 U dL^?1; FXIII, 31 U dL^?1. Moreover, coagulation factor activity levels that corresponded with Grade III bleeding were: undetectable levels for fibrinogen, FV and FXIII, < 15 U dL^?1 for combined FV + VIII; < 8 U dL^?1 for FVI; < 10 U dL^?1 for FX; and < 25 U dL^?1 for FXI. Conclusions: There is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in different RBDs. A strong association is only observed in fibrinogen, FX and FXIII deficiencies.     

In vitro assessment of bee venom effects on matrix metalloproteinase activity and interferon production

Controversial immunomodulatory properties of bee venom (BV) have provided an appropriate field for more investigation. The aim of present research was to verify the effects of honeybee venom on matrix metalloproteinase activity and interferon production as well as cell proliferation in monocyte and fibroblast cell lines.The monocyte and fibroblast cell lines (K562, HT-1080, WEHI-164) were used in order to assess proliferative response, interferon-1 production and matrix metalloproteinase-2 (MMP-2) activity. Australian BV (ABV) and Iranian BV (IBV) preparations at concentrations of 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 1microg/ml were added to each overnight cultured cells. In time course study, cells were treated each with ABV and IBV. In all cases supernatants were collected 24 hours after treatment. A sample of the each medium was used for zymography and interferons assay. Non-treated cells were used as controls.The production of IFN-a and IFN-b in supernatant of cell cultures was assessed using enzyme linked immunoassay procedure. MMP-2 activity, as an inflammatory index, was evaluated using zymoanalysis method.The results of this study showed that, there were no significant difference between two sources of honey bee venoms when they were added to an identical cell line, whereas, the responses of various cell lines against bee venom were different. The increasing amounts of bee venom to human monocyte cell line (K562) revealed a significant increase in proliferative response. Our findings showed that the bee venom had no influence on IFN-a production in cell culture media, whereas, adding the BV to K562 cell line could significantly increase the production level of IFN-b only on day 8 post-treatment. In addition the effect of bee venom on MMP-2 activity in both cell culture media, WEHI-164 and K562 was similar. The stimulatory effect of bee venom on MMP-2 activity occurred at low doses. In contrast, its inhibitory effect was seen at high concentrations.It is concluded that, honeybee venom affects on MMP-2 activity and interferon beta production as well as cell proliferation in a time and dose-dependent manner.