Downregulation of osteoblast markers and induction of the glial fibrillary acidic protein by oncostatin M in osteosarcoma cells require PKCdelta and STAT3.

The effects of OSM on proliferation and differentiation of osteosarcoma and nontransformed osteoblasts were analyzed. OSM downregulates osteoblast markers but induces the glial fibrillary acidic protein by the combined activation of PKCdelta and STAT3, offering new lines of therapeutic investigations. INTRODUCTION: Oncostatin M (OSM) is a multifunctional cytokine of the interleukin-6 family implicated in embryonic development, differentiation, inflammation, and regeneration of various tissues, mainly the liver, bone, and the central nervous and hematopoietic systems. One particularity of OSM relies on its growth inhibitory and pro-differentiating effects on a variety of tumor cell lines such as melanoma, providing arguments for a therapeutic application of OSM. The objective of this study was to analyze the effects of OSM on osteosarcoma cell lines proliferation and differentiation. MATERIALS AND METHODS: Proliferation was analyzed by 3H thymidine incorporation. Differentiation was analyzed by semiquantitative RT-PCR and immunocytochemistry for various markers. Alizarin red S staining was used to evaluate bone nodule formation. Morphological changes were studied by confocal and electron microscopy. Western blotting, kinases inhibitors, and dominant negative STAT3 were used to identified the signaling pathways implicated. RESULTS: OSM inhibits the growth of rat osteosarcoma cell lines as well as normal osteoblasts, in correlation with induction of the cyclin-dependent kinases inhibitor p21WAF1. However, OSM reduces osteoblast markers such as alkaline phosphatase, osteocalcin, and bone sialoprotein, leading to strong inhibition of mineralized nodule formation. This inhibitory effect is restricted to mature osteoblasts and differentiated osteosarcoma because OSM effectively stimulates osteoblast markers and bone nodule formation in early, but not late, bone marrow mesenchymal stem cell (BMSC) cultures. In osteosarcoma cells or BMSC, OSM induces expression of the glial fibrillary acidic protein (GFAP) as well as morphological and ultrastructural changes, for example, elongated shape and bundles of microfilaments in cell processes. Rottlerin (PKCdelta inhibitor), and to a lesser degree UO126 (MEK/ERK inhibitor), prevents the loss of osteoblastic markers by OSM, whereas dominant negative STAT3 prevents GFAP induction. CONCLUSIONS: These results highlight the particular gene expression profile of OSM-treated osteosarcoma cells and BMSCs, suggesting either a osteocytic or a glial-like phenotype. Together with the implication of PKCdelta, ERK1/2, and STAT3, these results offer new lines of investigations for neural cell transplantation and osteosarcoma therapy.     

Detection of the center of the hip joint in computer-assisted surgery: an evaluation study of the Surgetics algorithm.

OBJECTIVE: The aim of this paper is to assess the accuracy of an algorithm implemented by PRAXIM in the SURGETICS navigation station for detection of the hip center. This study will assess the robustness and accuracy of the algorithm in various clinical situations such as those involving non-sphericity of the femoral head, motion of the pelvis during hip center detection, and restricted range of motion. MATERIALS AND METHODS: The localization of the hip center, based on kinematics, relies on the recording of n successive positions of the femoral rigid body in the localizer reference system during a passive circumduction motion of the hip joint. Therefore, the shape of the clouds of points acquired may vary from one acquisition to the next. To allow a comprehensive study of the consequences of these variations for hip center detection, we developed a simulator to generate numerous clouds of points. Results given subsequently for each test are the values of the difference between the femoral mechanical axis computed with C(c), the computed hip center, and the same axis computed with C(o), the reference hip center. RESULTS: Test 1: Sensitivity to noise. The errors ranged from 3.33 E - 12 (SD 3.29E - 12) for a noise of 0 mm to 8.18E - 1 (SD - 7.05E - 1) for a noise of 15 mm. Test 2: Sensitivity to the shape of the acquisition motion. All trajectories gave an error < 1 degrees . Test 3: Sensitivity to restricted range of motion. No value > 1 degrees was found during this test. Test 4: Sensitivity to the distance between two points of the cloud. No value > 0.5 degrees was found during this test. Test 5: Sensitivity to the number of points included in the cloud. No value > 1 degrees was found during this test. CONCLUSIONS: The Surgetics algorithm is robust to noise, can compensate for pelvic motion, and can be used even in the case of restricted range of motion.     

Efficacy and safety of lowering immunosuppression to treat CMV infection in renal transplant recipients on valaciclovir prophylaxis: a pilot study.

BACKGROUND: Routine cytomegalovirus (CMV)-pp65 antigenaemia monitoring shows that some patients will develop pp65 antigenaemia during valaciclovir prophylaxis or after cessation of treatment. The aim of this pilot study was to evaluate the safety and efficacy of lowering immunosuppression in kidney transplant recipients who exhibit mildly symptomatic CMV infections while on valaciclovir prophylaxis. METHODS: We selected 12 patients who experienced mildly symptomatic CMV infections defined as a positive CMV-pp65 antigenaemia test associated with either neutropenia, asthenia or arthralgia, but no fever. All of them received prophylaxis with valaciclovir for at least 3 months. Testing for CMV-pp65 antigenaemia was performed weekly for 6 months. RESULTS: The mildly symptomatic infections occurred at a median interval of 69 days after transplantation-during prophylaxis in eight cases and after valaciclovir discontinuation in the other four cases. All of them were effectively managed by lowering immunosuppressive therapy, leading to the disappearance of symptoms and CMV antigenaemia reduction. No immunological complication or recurrence of CMV infection or disease was noted. I.v. ganciclovir never became necessary. CONCLUSION: The mildly symptomatic CMV infections occurring in valaciclovir-treated patients may be managed efficiently and without immunologic complication by lowering immunosuppressive therapy.     

Vectorization of morpholino oligomers by the (R-Ahx-R)4 peptide allows efficient splicing correction in the absence of endosomolytic agents.

The efficient and non-toxic nuclear delivery of steric-block oligonucleotides (ON) is a prerequisite for therapeutic strategies involving splice correction or exon skipping. Cationic cell penetrating peptides (CPPs) have given rise to much interest for the intracellular delivery of biomolecules, but their efficiency in promoting cytoplasmic or nuclear delivery of oligonucleotides has been hampered by endocytic sequestration and subsequent degradation of most internalized material in endocytic compartments. In the present study, we compared the splice correction activity of three different CPPs conjugated to PMO(705), a steric-block ON targeted against the mutated splicing site of human beta-globin pre-mRNA in the HeLa pLuc705 splice correction model. In contrast to Tat48-60 (Tat) and oligoarginine (R(9)F(2)) PMO(705) conjugates, the 6-aminohexanoic-spaced oligoarginine (R-Ahx-R)(4)-PMO(705) conjugate was able to promote an efficient splice correction in the absence of endosomolytic agents. Our mechanistic investigations about its uptake mechanisms lead to the conclusion that these three vectors are internalized using the same endocytic route involving proteoglycans, but that the (R-Ahx-R)(4)-PMO(705) conjugate has the unique ability to escape from lysosomial fate and to access to the nuclear compartment. This vector, which has displays an extremely low cytotoxicity, the ability to function without chloroquine adjunction and in the presence of serum proteins. It thus offers a promising lead for the development of vectors able to enhance the delivery of therapeutic steric-block ON in clinically relevant models.     

The nocturnal jejunal migrating motor complex: defining normal ranges by study of 51 healthy adult volunteers and meta-analysis

Interdigestive human small bowel motility is characterized by the migrating motor complex (MMC). The aims of this study were to: (i) establish the normal range of variables of the nocturnal jejunal MMC and (ii) incorporate these data in a subsequent meta-analysis. Eighty-one recordings were performed by prolonged (24 h) ambulatory manometry in 51 subjects in two centres. Quantitative analysis was undertaken of 419 Phase III and 332 Phase II episodes. Adjusted mean values of seven variables were calculated using a mixed-effects model. Meta-analysis of pooled published data to generate a reliable 95% reference range was also performed. Adjusted mean values and confidence intervals are presented for all seven variables. Intrasubject variances were large in comparison with intersubject. Meta-analysis of 19 studies (356 pooled patients) meeting inclusion criteria produced wide reference ranges. At least five such ranges are useful for the detection of abnormality in the individual. This is the largest study of normal volunteers presented to date, with ranges for many variables produced using appropriate statistical methodology. A model for definition of abnormality has been proposed. We recommend that these data may be used by investigators in this field as a complement to other existing indicators of small bowel dysmotility.     

Effect of female sex hormone supplementation and withdrawal on gastrointestinal and colonic transit in postmenopausal women

Females are disproportionately affected by constipation, which is often aggravated during pregnancy. Bowel function also changes during the luteal phase of the menstrual cycle. The aim was to compare the effects of acute administration of female sex steroids on gastric emptying, small bowel transit and colonic transit in healthy postmenopausal subjects. A second aim was to determine whether withdrawal of the hormones was associated with a change in transit. Forty-nine postmenopausal females were randomized to receive for 7 days 400 mg day(-1) micronized progesterone, 0.2 mg day(-1) oestradiol, combination of the two, or placebo. Treatment groups were balanced on age. Participants underwent whole gut transit measurement by scintigraphy using a 99m-labeled technetium-egg meal and 111-labeled indium-charcoal via a delayed-release capsule. Transit measurement was repeated after withdrawal of the study medications. The primary endpoints were ascending colon (AC) emptying half-life time (t1/2) and colonic geometric centre (GC) at 24 h. Secondary analysis variables were GC at 4 and 48 h, gastric emptying t1/2 and colonic filling at 6 h. There was a significant overall effect of progesterone on colonic transit with shorter AC emptying t1/2 and significantly greater colonic GC at 48 h. No transit endpoints were altered by oestradiol or combined hormonal treatment relative to placebo. Oestradiol and progesterone resulted in looser stool consistency. Withdrawal of the hormone supplement was not associated with significant alteration in transit. Micronized progesterone does not retard colonic transit in postmenopausal females.