Effects of rat anti-VEGF antibody in a rat model of corneal graft rejection by topical and subconjunctival routes.

PURPOSE: To compare the effect of a rat anti-VEGF antibody, administered either by topical or subconjunctival (SC) routes, on a rat model of corneal transplant rejection.METHODS: Twenty-four rats underwent corneal transplantation and were randomized into four treatment groups (n=6 in each group). G1 and G2 received six SC injections (0.02 ml 10 µg/ml) of denatured (G1) or active (G2) anti-VEGF from Day 0 to Day 21 every third day. G3 and G4 were instilled three times a day with denatured (G3) or active (G4) anti-VEGF drops (10 µg/ml) from Day 0 to Day 21. Corneal mean clinical scores (MCSs) of edema (E), transparency (T), and neovessels (nv) were recorded at Days 3, 9, 15, and 21. Quantification of neovessels was performed after lectin staining of vessels on flat mounted corneas.RESULTS: Twenty-one days after surgery, MCSs differed significantly between G1 and G2, but not between G3 and G4, and the rejection rate was significantly reduced in rats receiving active antibodies regardless of the route of administration (G2=50%, G4=66.65% versus G1 and G3=100%; p<0.05). The mean surfaces of neovessels were significantly reduced in groups treated with active anti-VEGF (G2, G4). However, anti-VEGF therapy did not completely suppress corneal neovessels.CONCLUSIONS: Specific rat anti-VEGF antibodies significantly reduced neovascularization and subsequent corneal graft rejection. The SC administration of the anti-VEGF antibody was more effective than topical instillation.

Administration of Il-18bp by Gene Therapy Reduces Inflammation and Prevents Joint Destruction by Downregulation of Mmp9 In Rat Aia: Role ff Mmp9 In Bone and Joint Destruction in Arthritis

Background and objectives Interleukin 18 (IL-18) is a pleiotropic cytokine involved in rheumatoid arthritis (RA) pathogenesis. This study was carried out to evaluate the effi cacy of IL-18 binding protein (IL-18BP) gene therapy in the rat adjuvant- induced arthritis (AIA) model and to decipher the mechanisms by which IL-18BP delivery lessens bone destruction.Materials and methods Arthritis was induced in female Lewis rat by Mycobacterium butyricum and the mRNA expression of IL-18 and IL-18BP was determined in the joints. In a preventive study, rats were divided into an adenovirus producing IL-18BP-Fc (AdmIL-18BP-Fc) group (n=8) and an adenovirus producing green fl uorescent protein (AdGFP) group (n=7). On day 8 after AIA induction, adenoviruses were injected. Clinical parameters were assessed. At day 18, during maximal arthritis, the rats were euthanized, ankles were collected and x-rays were performed. mRNA and protein were extracted from joints for analysis by quantitative reverse transcriptase-PCR, multiplex, Western blot and zymography.Results The authors observed a decrease in the (IL-18BP/ IL-18) ratio from day 7 to 45. Administration of AdmIL-18BPd-Fc decreased clinical parameters and prevented bone and joint destruction compared to AdGFP administration. IL-18BP delivery reduced the (receptor activator of nuclear factor κB ligand (RANKL)/osteoprotegerin (OPG)) ratio by 70%, the matrix metalloproteinase 9 (MMP9) level by 33% and the tartrate-resistant acid phosphatase (TRAP) level by 44% in the joint homogenates from AdmIL-18BPd-Fc compared to AdGFP treated rats.Conclusions In rat AIA, a decrease in the (IL-18BP/IL-18) ratio was observed. IL-18BP delivery prevented joint and bone destruction by downregulating MMP9, (RANKL/OPG) and TRAP, suggesting a potential benefi t of a similar therapy in RA.Abstract topics Towards novel therapeutic strategies.

European French-speaking study from the GEMO group on bone metastases management: a special focus on external beam radiotherapy practice survey.

This study seeks to perform a survey of patterns of practice among the different physicians involved in the bone metastases management, with special focus on external beam radiotherapy (EBRT).A questionnaire about bone metastases based on clinical cases and supplemented with general questions, including medical therapies, EBRT and metabolic radiotherapy strategies, surgery, and supportive care approaches, was sent to 4,706 French-speaking physicians in Belgium, France, Luxemburg, and Switzerland.Overall, 644 questionnaires were analyzed. Twenty-eight percent concerned the radiotherapy approach and were judged adequate to respond to the part dedicated to EBRT. Sixty-nine percent of physicians used a total dose irradiation of 30 Gy delivered in ten fractions. A large majority (75%) used two opposed fields prescribed at mid-depth (30%), or with non-equally weighted fields (45%). Seventy percent irradiated also above and below the concerned vertebra. A dosimetry planning treatment was done in 85% and high-energy megavoltage photons were used in 42%. Moreover, 54% physicians used short course radiotherapy in routine.Radiotherapy remains the mainstay of treatment of bone metastases, but there is substantial heterogeneity in clinical practice. Guidelines and treatment protocols are required to improve the treatment quality.

Trabecular bone score: a noninvasive analytical method based upon the DXA image.

The trabecular bone score (TBS) is a gray-level textural metric that can be extracted from the two-dimensional lumbar spine dual-energy X-ray absorptiometry (DXA) image. TBS is related to bone microarchitecture and provides skeletal information that is not captured from the standard bone mineral density (BMD) measurement. Based on experimental variograms of the projected DXA image, TBS has the potential to discern differences between DXA scans that show similar BMD measurements. An elevated TBS value correlates with better skeletal microstructure; a low TBS value correlates with weaker skeletal microstructure. Lumbar spine TBS has been evaluated in cross-sectional and longitudinal studies. The following conclusions are based upon publications reviewed in this article: 1) TBS gives lower values in postmenopausal women and in men with previous fragility fractures than their nonfractured counterparts; 2) TBS is complementary to data available by lumbar spine DXA measurements; 3) TBS results are lower in women who have sustained a fragility fracture but in whom DXA does not indicate osteoporosis or even osteopenia; 4) TBS predicts fracture risk as well as lumbar spine BMD measurements in postmenopausal women; 5) efficacious therapies for osteoporosis differ in the extent to which they influence the TBS; 6) TBS is associated with fracture risk in individuals with conditions related to reduced bone mass or bone quality. Based on these data, lumbar spine TBS holds promise as an emerging technology that could well become a valuable clinical tool in the diagnosis of osteoporosis and in fracture risk assessment.

Combining bone resorption markers and heel quantitative ultrasound to discriminate between fracture cases and control

Rapport de synthèse : L'ostéoporose est reconnue comme un problème majeur de santé publique. Comme il existe actuellement des traitements préventifs efficaces pour minimiser le risque de fracture, il est essentiel de développer des nouvelles stratégies de détection des femmes à risque de fracture. Les marqueurs spécifiques du remodelage osseux dosés dans les urines ainsi que les ultrasons quantitatifs du talon ont été étudiés comme outils cliniques pour prédire le risque fracturaire chez les femmes âgées. Il n'existe cependant que très peu de donnée sur la combinaison de ces deux outils pour améliorer la prédiction du risque de fracture. Cette étude cas-contrôle, réalisée chez 368 femmes âgées de 76 ans en moyenne d'une cohorte suisse de femmes ambulatoires, évalue la capacité discriminative entre 195 femmes avec fracture non-vertébrale à bas traumatisme et 173 femmes sans fractures - de deux marqueurs urinaires de la résorption osseuse, les pyridinolines et les deoxypyridinolines, ainsi que deux ultrasons quantitatifs du talon, le Achilles+ (GE-Lunar, Madison, USA) et le Sahara (Hologic, Waltham, USA). Les 195 patientes avec une fracture ont été choisies identiques aux 173 contrôles concernant Page, l'indice de masse corporel, le centre médical et la durée de suivi jusqu'à la fracture. Cette étude montre que les marqueurs urinaires de la résorption osseuse ont une capacité environ identique aux ultrasons quantitatifs du talon pour discriminer entre les patientes avec fracture non-vertébrale à bas traumatisme et les contrôles. La combinaison des deux tests n'est cependant pas plus performante qu'un seul test. Les résultats de cette étude peuvent aider à concevoir les futures stratégies de détection du risque fracturaire chez les femmes âgées, qui intègrent notamment des facteurs de risque cliniques, radiologiques et biochimiques. Abstract : Summary : This nested case-control analysis of a Swiss ambulatory cohort of elderly women assessed the discriminatory power of urinary markers of bone resorption and heel quantitative ultrasound for non-vertebral fractures. The tests all discriminated between cases and controls, but combining the two strategies yielded no additional relevant information. Introduction : Data are limited regarding the combination of bone resorption markers and heel quantitative bone ultrasound (QUS) in the detection of women at risk for fracture. Methods In a nested case-control analysis, we studied 368 women (mean age 76.213.2 years), 195 with low-trauma non-vertebral fractures and 173 without, matched for age, BMI, medical center, and follow-up duration, from a prospective study designed to predict fractures. Urinary total pyridinolines (PYD) and deoxypyridinolines (DPD) were measured by high performance liquid chromatography. All women underwent bone evaluations using Achilles+ and Sahara heel QUS. Results : Areas under the receiver operating-characteristic curve (AUC) for discriminative models of the fracture group, with 95% confidence intervals, were 0.62 (0.560.68) and 0.59 (0.53-0.65) for PYD and DPD, and 0.64 (0.58-0.69) and 0.65 (0.59-0.71) for Achilles+ and Sahara QUS, respectively. The combination of resorption markers and QUS added no significant discriminatory information to either measurement alone with an AUC of 0.66 (0.600.71) for Achilles+ with PYD and 0.68 (0.62-0.73) for Sahara with PYD. Conclusions : Urinary bone resorption markers and QUS are equally discriminatory between non-vertebral fracture patients and controls. However, the combination of bone resorption markers and QUS is not better than either test used alone.

TBS (trabecular bone score) and diabetes-related fracture risk.

CONTEXT: Type 2 diabetes is associated with increased fracture risk but paradoxically greater bone mineral density (BMD). Trabecular bone score (TBS) is derived from the texture of the spine dual x-ray absorptiometry (DXA) image and is related to bone microarchitecture and fracture risk, providing information independent of BMD. OBJECTIVE: This study evaluated the ability of lumbar spine TBS to account for increased fracture risk in diabetes. DESIGN AND SETTING: We performed a retrospective cohort study using BMD results from a large clinical registry for the province of Manitoba, Canada. Patients: We included 29,407 women 50 years old and older with baseline DXA examinations, among whom 2356 had diagnosed diabetes. MAIN OUTCOME MEASURES: Lumbar spine TBS was derived for each spine DXA examination blinded to clinical parameters and outcomes. Health service records were assessed for incident nontraumatic major osteoporotic fractures (mean follow-up 4.7 years). RESULTS: Diabetes was associated with higher BMD at all sites but lower lumbar spine TBS in unadjusted and adjusted models (all P < .001). The adjusted odds ratio (aOR) for a measurement in the lowest vs the highest tertile was less than 1 for BMD (all P < .001) but was increased for lumbar spine TBS [aOR 2.61, 95% confidence interval (CI) 2.30-2.97]. Major osteoporotic fractures were identified in 175 women (7.4%) with and 1493 (5.5%) without diabetes (P < .001). Lumbar spine TBS was a BMD-independent predictor of fracture and predicted fractures in those with diabetes (adjusted hazard ratio 1.27, 95% CI 1.10-1.46) and without diabetes (hazard ratio 1.31, 95% CI 1.24-1.38). The effect of diabetes on fracture was reduced when lumbar spine TBS was added to a prediction model but was paradoxically increased from adding BMD measurements. CONCLUSIONS: Lumbar spine TBS predicts osteoporotic fractures in those with diabetes, and captures a larger portion of the diabetes-associated fracture risk than BMD.

Staphylococcal biofilm formation on the surface of three different calcium phosphate bone grafts: a qualitative and quantitative in vivo analysis.

Differences in physico-chemical characteristics of bone grafts to fill bone defects have been demonstrated to influence in vitro bacterial biofilm formation. Aim of the study was to investigate in vivo staphylococcal biofilm formation on different calcium phosphate bone substitutes. A foreign-body guinea-pig infection model was used. Teflon cages prefilled with β-tricalcium phosphate, calcium-deficient hydroxyapatite, or dicalcium phosphate (DCP) scaffold were implanted subcutaneously. Scaffolds were infected with 2 × 10(3) colony-forming unit of Staphylococcus aureus (two strains) or S. epidermidis and explanted after 3, 24 or 72 h of biofilm formation. Quantitative and qualitative biofilm analysis was performed by sonication followed by viable counts, and microcalorimetry, respectively. Independently of the material, S. aureus formed increasing amounts of biofilm on the surface of all scaffolds over time as determined by both methods. For S. epidermidis, the biofilm amount decreased over time, and no biofilm was detected by microcalorimetry on the DCP scaffolds after 72 h of infection. However, when using a higher S. epidermidis inoculum, increasing amounts of biofilm were formed on all scaffolds as determined by microcalorimetry. No significant variation in staphylococcal in vivo biofilm formation was observed between the different materials tested. This study highlights the importance of in vivo studies, in addition to in vitro studies, when investigating biofilm formation of bone grafts.

Cytomegalovirus serology and replication remain associated with solid organ graft rejection and graft loss in the era of prophylactic treatment.

BACKGROUND: Cytomegalovirus (CMV) replication has been associated with more risk for solid organ graft rejection. We wondered whether this association still holds when patients at risk receive prophylactic treatment for CMV. METHODS: We correlated CMV infection, biopsy-proven graft rejection, and graft loss in 1,414 patients receiving heart (n=97), kidney (n=917), liver (n=237), or lung (n=163) allografts reported to the Swiss Transplant Cohort Study. RESULTS: Recipients of all organs were at an increased risk for biopsy-proven graft rejection within 4 weeks after detection of CMV replication (hazard ratio [HR] after heart transplantation, 2.60; 95% confidence interval [CI], 1.34-4.94, P<0.001; HR after kidney transplantation, 1.58; 95% CI, 1.16-2.16, P=0.02; HR after liver transplantation, 2.21; 95% CI, 1.53-3.17, P<0.001; HR after lung transplantation, 5.83; 95% CI, 3.12-10.9, P<0.001. Relative hazards were comparable in patients with asymptomatic or symptomatic CMV infection. The CMV donor or recipient serological constellation also predicted the incidence of graft rejection after liver and lung transplantation, with significantly higher rates of rejection in transplants in which donor or recipient were CMV seropositive (non-D-/R-), compared with D- transplant or R- transplant (HR, 3.05; P=0.002 for liver and HR, 2.42; P=0.01 for lung transplants). Finally, graft loss occurred more frequently in non-D- or non-R- compared with D- transplant or R- transplant in all organs analyzed. Valganciclovir prophylactic treatment seemed to delay, but not prevent, graft loss in non-D- or non-R- transplants. CONCLUSION: Cytomegalovirus replication and donor or recipient seroconstellation remains associated with graft rejection and graft loss in the era of prophylactic CMV treatment.

Nouvelles thérapies pour les maladies osseuses de l'enfant [New therapies for children affected by bone diseases].

Considerable progress has been achieved in recent years in treating children affected by bone diseases. Advances in the understanding of the molecular pathophysiology of genetic bone diseases have led to the development of enzyme replacement therapies for various lysosomal storage diseases, following the breakthrough initiated in treating Gaucher disease. Clinical studies are underway with tailored molecules correcting bone fragility and alleviating chronic bone pain and other manifestations of hypophosphatasia, or promoting growth of long bones in achondroplasia patients. We further report our very encouraging experience with intravenous bisphosphonate treatment in children suffering from secondary osteopenia and the high prevalence of calcium and vitamin D deficits in these severely disabled children.

Alterations of bone microarchitecture in young patients with inflammatory bowel diseases are associated with fracture risk during growth

Aims: Inflammatory bowel diseases (IBD) appearing during childhood and adolescence compromise peak bone mass acquisition and increase fracture risk. The structural determinants of bone fragility in IBD however remain unknown. Methods: We investigated volumetric bone mineral density (vBMD), trabecular and cortical bone microstructure at distal radius and tibia by high-resolution pQCT (XtremeCT, Scanco, Switzerland), aBMD at distal radius, hip and spine and vertebral fracture assessment (VFA) by DXA in 107 young patients (mean age 22.8 yrs, range 12.2-33.7 yrs; 62 females and 45 males) with Crohn's disease (n=75), ulcerative colitis (n=25), undetermined colitis (n=2), and no definitive diagnosis (n=5), and in 389 healthy young individuals. Results: Mean disease duration was 6.1 yrs, 89/107 IBD patients received corticosteroids, 83 other immunomodulators, and 59 vitamin D. Clinical fractures were reported by 38 patients (mean age at 1st fracture, 12.6 yrs), the vast majority of the forearm, arm or hand; 5 had vertebral crush fractures (Grade 1 or 2) and 11 had multiple fractures. As compared to healthy controls (matched 2:1 for age, sex, height and fracture history), the 102 patients with established IBD had similar weight but significantly lower aBMD at all sites, lower trabecular (Tb) BV/TV and number, and greater Tb separation and inhomogeneous Tb distribution (1/SD TbN) at both distal radius and tibia, lower tibia cortical thickness (CTh), but no differences in cortical vBMD nor bone perimeter. Among IBD's, aBMD was not associated with fractures (by logistic regression adjusted for age, age square, sex, height, weight and protein intake). However, radius and tibia Tb BV/TV, thickness and SD 1/TbN, as well as radius Tb separation and perimeter, were significantly associated with fracture risk (fully adjusted as above), whereas cortical vBMD and CTh were not. After adjustment for aBMD at radius, respectively at femur neck, radius SD 1/TbN and tibia BV/TV, TbTh and perimeter remained independently associated with fracture risk. Conclusions: Young subjects with IBD have low bone mass and poor bone microarchitecture compared to healthy controls. Alterations of bone microarchitecture, particularly in the trabecular bone compartment, are specifically associated with increased fracture risk during growth.

Trabecular bone score is associated with volumetric bone density and microarchitecture as assessed by central QCT and HRpQCT in Chinese American and white women.

Although high-resolution peripheral quantitative computed tomography (HRpQCT) and central quantitative computed tomography (QCT) studies have shown bone structural differences between Chinese American (CH) and white (WH) women, these techniques are not readily available in the clinical setting. The trabecular bone score (TBS) estimates trabecular microarchitecture from dual-energy X-ray absorptiometry spine images. We assessed TBS in CH and WH women and investigated whether TBS is associated with QCT and HRpQCT indices. Areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry, lumbar spine (LS) TBS, QCT of the LS and hip, and HRpQCT of the radius and tibia were performed in 71 pre- (37 WH and 34 CH) and 44 postmenopausal (21 WH and 23 CH) women. TBS did not differ by race in either pre- or postmenopausal women. In the entire cohort, TBS positively correlated with LS trabecular volumetric bone mineral density (vBMD) (r = 0.664), femoral neck integral (r = 0.651), trabecular (r = 0.641) and cortical vBMD (r = 0.346), and cortical thickness (C/I; r = 0.540) by QCT (p < 0.001 for all). TBS also correlated with integral (r = 0.643), trabecular (r = 0.574) and cortical vBMD (r = 0.491), and C/I (r = 0.541) at the total hip (p < 0.001 for all). The combination of TBS and LS aBMD predicted more of the variance in QCT measures than aBMD alone. TBS was associated with all HRpQCT indices (r = 0.20-0.52) except radial cortical thickness and tibial trabecular thickness. Significant associations between TBS and measures of HRpQCT and QCT in WH and CH pre- and postmenopausal women demonstrated here suggest that TBS may be a useful adjunct to aBMD for assessing bone quality.

Early Stage Primary Bone Lymphoma: A Retrospective, Multicenter Rare Cancer Network Study

Purpose/Objective(s): Primary bone lymphoma (PBL) represents less than 1% of all malignant lymphomas, and 4-5% of all extranodal lymphomas. In this study, we assessed the disease profile, outcome, and prognostic factors in patients with stage I and II PBL.Materials/Methods: Between 1987 and 2008, 116 consecutive patients with PBL treated in 13 RCNinstitutions were included in this study. Inclusion criteriawere: age.17 yrs, PBLin stage I and II, andminimum6months follow-up. The median agewas 51 yrs (range: 17-93).Diagnosticwork-up included plain boneXray (74%of patients), scintigraphy (62%), CT-scan (65%),MRI (58%), PET (18%), and bone-marrow biopsy (84%).All patients had biopsy-proven confirmation of non-Hodgkin's lymphoma (NHL). The histopathological type was predominantly diffuse large B-cell lymphoma (78%) and follicular lymphoma (6%), according to theWHOclassification. One hundred patients had a high-grade, 7 intermediate and 9 low-gradeNHL. Ninety-three patients had anAnn-Arbor stage I, and 23 had a stage II. Seventy-seven patients underwent chemoradiotherapy (CXRT), 12 radiotherapy (RT) alone, 10 chemotherapy alone (CXT), 9 surgery followed by CXRT, 5 surgery followed by CXT, and 2 surgery followed by RT. One patient died before treatment.Median RT dosewas 40Gy (range: 4-60).Themedian number ofCXTcycleswas 6 (range, : 2-8).Median follow-upwas 41months (range: 6-242).Results: Following treatment, the overall response rate was 91% (CR 74%, PR 17%). Local recurrence was observed in 12 (10%) patients, and systemic recurrence in 17 (15%) patients. Causes of death included disease progression in 16, unrelated disease in 6, CXT-related toxicity in 1, and secondary cancer in 2 patients. The 5-yr overall survival (OS), disease-free survival (DFS), lymphoma- specific survival (LSS), and local control (LC) were 76%, 69%, 78%, and 92%, respectively. In univariate analyses (log-rank test), favorable prognostic factors for survival were: age\50 years (p = 0.008), IPI score #1 (p = 0.009), complete response (p\0.001), CXT (p = 0.008), number of CXT cycles $6 (p = 0.007), and RT dose . 40 Gy (p = 0.005). In multivariate analysis age, RT dose, complete response, and absence of B symptoms were independent factors influencing the outcome. There were 3 patients developing grade 3 or more (CTCAE.V3.0) toxicities.Conclusions: This large multicenter study, confirms the relatively good prognosis of early stage PBL, treated with combined CXRT. Local control was excellent, and systemic failure occurred infrequently. A sufficient dose of RT (. 40 Gy) and completeCXT regime (. 6 cycles) were associated with a better outcome. Combined modality appears to be the treatment of choice.Author Disclosure: L. Cai, None; M.C. Stauder, None; Y.J. Zhang, None; P. Poortmans, None; Y.X. Li, None; N. Constantinou, None; J. Thariat, None; S. Kadish, None; M. Ozsahin, None; R.O. Mirimanoff, None.

PPARβ/δ governs Wnt signaling and bone turnover.

Peroxisome proliferator-activated receptors (PPARs) act as metabolic sensors and central regulators of fat and glucose homeostasis. Furthermore, PPARγ has been implicated as major catabolic regulator of bone mass in mice and humans. However, a potential involvement of other PPAR subtypes in the regulation of bone homeostasis has remained elusive. Here we report a previously unrecognized role of PPARβ/δ as a key regulator of bone turnover and the crosstalk between osteoblasts and osteoclasts. In contrast to activation of PPARγ, activation of PPARβ/δ amplified Wnt-dependent and β-catenin-dependent signaling and gene expression in osteoblasts, resulting in increased expression of osteoprotegerin (OPG) and attenuation of osteoblast-mediated osteoclastogenesis. Accordingly, PPARβ/δ-deficient mice had lower Wnt signaling activity, lower serum concentrations of OPG, higher numbers of osteoclasts and osteopenia. Pharmacological activation of PPARβ/δ in a mouse model of postmenopausal osteoporosis led to normalization of the altered ratio of tumor necrosis factor superfamily, member 11 (RANKL, also called TNFSF11) to OPG, a rebalancing of bone turnover and the restoration of normal bone density. Our findings identify PPARβ/δ as a promising target for an alternative approach in the treatment of osteoporosis and related diseases.