Association between PET/CT Scan Findings, Treatment, and Cancer Incidence in a Cohort of AAA Patients.

Background: Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease that poses several challenges. Given the increasing evidence that AAA patients are more likely to develop cancer and the importance of its early detection, we strived to develop a non-invasive tool based on serial FDG-PET/CT scan examinations to identify, among AAA patients, those at risk of cancer. Methods: Between 2006 and 2011 we recruited 149 AAA patients, free of cancer at baseline, and followed them until the end of 2021. All patients underwent an FDG-PET/CT scan at inclusion and possibly more scans during follow-up. At each medical imaging examination, the aneurysmal FDG uptake was recorded. Patients were stratified based on their aortic wall PET status (negative/positive). Any occurrence of cancer was reported. A Cox regression analysis and competing-risk modeling were applied to the data. Results: The proportion of AAA patients who developed cancer was 31.5% (mean time to diagnosis was 5.7 ± 3.4 years) and the death rate was 59%. A difference in cancer incidence between PET+ and PET- patients was detected (46.8% vs. 27.3%; HR = 1.96, 95%CI: 1.07-3.57, p = 0.028). Moreover, AAA patients undergoing surgical treatment had a lower risk of cancer than unoperated patients (28% vs. 50%; HR = 0.41, 95%CI: 0.21-0.80, p = 0.009). Conclusions: In AAA patients, diagnostic imaging with an FDG-PET/CT scan can help identify those patients at a higher risk of developing cancer. Moreover, the higher cancer risk in non-surgically treated patients calls for further analysis of associations between aneurysm growth and malignant disease.

Structural and functional heterogeneity of mineralized fibrocartilage at the Achilles tendon-bone insertion.

A demanding task of the musculoskeletal system is the attachment of tendon to bone at entheses. This region often presents a thin layer of fibrocartilage (FC), mineralized close to the bone and unmineralized close to the tendon. Mineralized FC deserves increased attention, owing to its crucial anchoring task and involvement in enthesis pathologies. Here, we analyzed mineralized FC and subchondral bone at the Achilles tendon-bone insertion of rats. This location features enthesis FC anchoring tendon to bone and sustaining tensile loads, and periosteal FC facilitating bone-tendon sliding with accompanying compressive and shear forces. Using a correlative multimodal investigation, we evaluated potential specificities in mineral content, fiber organization and mechanical properties of enthesis and periosteal FC. Both tissues had a lower degree of mineralization than subchondral bone, yet used the available mineral very efficiently: for the same local mineral content, they had higher stiffness and hardness than bone. We found that enthesis FC was characterized by highly aligned mineralized collagen fibers even far away from the attachment region, whereas periosteal FC had a rich variety of fiber arrangements. Except for an initial steep spatial gradient between unmineralized and mineralized FC, local mechanical properties were surprisingly uniform inside enthesis FC while a modulation in stiffness, independent from mineral content, was observed in periosteal FC. We interpreted these different structure-property relationships as a demonstration of the high versatility of FC, providing high strength at the insertion (to resist tensile loading) and a gradual compliance at the periosteal surface (to resist contact stresses). STATEMENT OF SIGNIFICANCE: Mineralized fibrocartilage (FC) at entheses facilitates the integration of tendon in bone, two strongly dissimilar tissues. We focus on the structure-function relationships of two types of mineralized FC, enthesis and periosteal, which have clearly distinct mechanical demands. By investigating them with multiple high-resolution methods in a correlative manner, we demonstrate differences in fiber architecture and mechanical properties between the two tissues, indicative of their mechanical roles. Our results are relevant both from a medical viewpoint, targeting a clinically relevant location, as well as from a material science perspective, identifying FC as high-performance versatile composite.

Differential Biological Effects of Dietary Lipids and Irradiation on the Aorta, Aortic Valve, and the Mitral Valve.

Aims: Dietary cholesterol and palmitic acid are risk factors for cardiovascular diseases (CVDs) affecting the arteries and the heart valves. The ionizing radiation that is frequently used as an anticancer treatment promotes CVD. The specific pathophysiology of these distinct disease manifestations is poorly understood. We, therefore, studied the biological effects of these dietary lipids and their cardiac irradiation on the arteries and the heart valves in the rabbit models of CVD. Methods and Results: Cholesterol-enriched diet led to the thickening of the aortic wall and the aortic valve leaflets, immune cell infiltration in the aorta, mitral and aortic valves, as well as aortic valve calcification. Numerous cells expressing α-smooth muscle actin were detected in both the mitral and aortic valves. Lard-enriched diet induced massive aorta and aortic valve calcification, with no detectable immune cell infiltration. The addition of cardiac irradiation to the cholesterol diet yielded more calcification and more immune cell infiltrates in the atheroma and the aortic valve than cholesterol alone. RNA sequencing (RNAseq) analyses of aorta and heart valves revealed that a cholesterol-enriched diet mainly triggered inflammation-related biological processes in the aorta, aortic and mitral valves, which was further enhanced by cardiac irradiation. Lard-enriched diet rather affected calcification- and muscle-related processes in the aorta and aortic valve, respectively. Neutrophil count and systemic levels of platelet factor 4 and ent-8-iso-15(S)-PGF2α were identified as early biomarkers of cholesterol-induced tissue alterations, while cardiac irradiation resulted in elevated levels of circulating nucleosomes. Conclusion: Dietary cholesterol, palmitic acid, and cardiac irradiation combined with a cholesterol-rich diet led to the development of distinct vascular and valvular lesions and changes in the circulating biomarkers. Hence, our study highlights unprecedented specificities related to common risk factors that underlie CVD.

Local anisotropy in mineralized fibrocartilage and subchondral bone beneath the tendon-bone interface.

The enthesis allows the insertion of tendon into bone thanks to several remarkable strategies. This complex and clinically relevant location often features a thin layer of fibrocartilage sandwiched between tendon and bone to cope with a highly heterogeneous mechanical environment. The main purpose of this study was to investigate whether mineralized fibrocartilage and bone close to the enthesis show distinctive three-dimensional microstructural features, possibly to enable load transfer from tendon to bone. As a model, the Achilles tendon-calcaneus bone system of adult rats was investigated with histology, backscattered electron imaging and micro-computed tomography. The microstructural porosity of bone and mineralized fibrocartilage in different locations including enthesis fibrocartilage, periosteal fibrocartilage and bone away from the enthesis was characterized. We showed that calcaneus bone presents a dedicated protrusion of low porosity where the tendon inserts. A spatially resolved analysis of the trabecular network suggests that such protrusion may promote force flow from the tendon to the plantar ligament, while partially relieving the trabecular bone from such a task. Focusing on the tuberosity, highly specific microstructural aspects were highlighted. Firstly, the interface between mineralized and unmineralized fibrocartilage showed the highest roughness at the tuberosity, possibly to increase failure resistance of a region carrying large stresses. Secondly, fibrochondrocyte lacunae inside mineralized fibrocartilage, in analogy with osteocyte lacunae in bone, had a predominant alignment at the enthesis and a rather random organization away from it. Finally, the network of subchondral channels inside the tuberosity was highly anisotropic when compared to contiguous regions. This dual anisotropy of subchondral channels and cell lacunae at the insertion may reflect the alignment of the underlying collagen network. Our findings suggest that the microstructure of fibrocartilage may be linked with the loading environment. Future studies should characterize those microstructural aspects in aged and or diseased conditions to elucidate the poorly understood role of bone and fibrocartilage in enthesis-related pathologies.

Comprehensive analysis of the immunomodulatory effects of rapamycin on human T cells in graft-versus-host disease prophylaxis.

Graft-versus-host disease (GVHD) is a major cause of toxicity after allogeneic hematopoietic cell transplantation (allo-HCT). While rapamycin (RAPA) is commonly used in GVHD prophylaxis in combination with a calcineurin inhibitor (CNI), the understanding of its mechanism of action on human T cells is still incomplete. Here, we performed an extensive analysis of RAPA effects on human T cells in a humanized mouse model of GVHD, in ex-vivo T cell cultures and in patients given RAPA plus tacrolimus as GVHD prophylaxis after nonmyeloablative allo-HCT. We demonstrate that RAPA mitigates GVHD by decreasing T cell engraftment and differentiation, inhibiting CD8+ T cell activation and increasing the long-term IL-2 secretion, thereby supporting regulatory T cell (Treg) proliferation. In contrast, graft-versus-leukemia effects were not abrogated, as RAPA-treated T cells had increased resistance to apoptosis and retained their effector function and proliferative capacity upon re-stimulation. Importantly, we found that RAPA impact on Treg and CD8+ T cells was closely dependent upon IL-2 signaling and that therapeutic options interfering with IL-2, such as calcineurin inhibitors, antagonize the IL-2-dependent promotion of Treg mediated by RAPA. Our results suggest that RAPA immunological efficacy could be improved in combination with drugs having possible synergistic effects such as the hypomethylating agent 5-azacytidine.

Practical considerations for reducing mortality rates in alloxan-induced diabetic rabbits.

Chemically-induced diabetic animal models have been employed in many areas of diabetes mellitus (DM) research, but managing post-induction animal survival rates remains one of the main downsides. The aim of the present study was to propose a reliable approach to animal management and monitoring after DM induction in a rabbit model in order to reduce animal mortality rates. DM was induced by injecting alloxan in 12 New Zealand White rabbits. A preventive subcutaneous glucose administration to counteract a potentially lethal hypoglycemic phase following alloxan injection was performed on individual bases. Blood glucose level (BGL) was checked hourly for the first 36 h, then every 2 h until the hyperglycemic state was confirmed. All 12 rabbits survived a 48-hour post-induction phase. The critical hypoglycemic phase's start points and duration differed significantly among the rabbits, lasting from 6.7 to 37 h (19.75 ± 8.44). The rabbits entered the final hyperglycemic phase 18 h at the earliest and 42 h at the latest after induction (26.63 ± 7.07). The average daily BGLs throughout the study period ranged from 268 to 512 mg/dL (413.73 ± 76.69). Eleven rabbits survived until the end of the experiment. The variability of rabbits' responses to alloxan injection emphasizes the importance of monitoring rabbit behavior and thoroughly checking BGLs, followed by a preventive glucose administration based on rabbits' individual needs for up to 36 h after alloxan injection. The proposed approach seems to reduce animal mortality.

Platelet-rich plasma (PRP) and tendon healing: comparison between fresh and frozen-thawed PRP.

Platelet-rich plasma (PRP) is increasingly used in the treatment of musculoskeletal diseases. Its preservation by freezing it for the realization of multiple injections in clinical use has never been discussed. Calcaneal tendons of rats were surgically sectioned. Platelet concentration of the PRP was 2.5 x 106/µl with autologous plasma of rats. Frozen-thawed PRP was prepared by performing two cycles of freezing and thawing on PRP aliquots. Both platelet preparations were injected in the lesion. Biomechanical and histological evaluations were carried out after 7, 20 or 40 days post surgery. After 7 and 40 days, no significant difference was observed between the PRP and the frozen-thawed PRP group. There is however a difference 20 days after surgery: the ultimate tensile strength (UTS) was greater in the fresh PRP group. No obvious difference with histological aspect was observed between the two groups. In conclusion, fresh PRP and frozen-thawed PRP injections can lead to similar results in the healing process of section calcaneal tendons of rats. Improvements with fresh PRP are slight. PRP could thus be frozen to be preserved if multiple injections are needed (e.g. osteoarthritis).

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description.

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily. Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Therapeutic Applications of Prostaglandins and Thromboxane A2 Inhibitors in Abdominal Aortic Aneurysms.

BACKGROUND: Abdominal aortic aneurysm (AAA) is one of the leading causes of death in western countries. Surgery is still, at the present time, the sole treatment that has however a significant mortality and cost rate. Many pharmacological agents are under investigation aiming to reduce growth and prevent AAA rupture. These drugs target different pathological pathways and, notably, the excessive production of prostanoids by cyclooxygenases (COX). Intra-aneurysmal thrombus plays an adverse key role in the progression of AAA, platelets being a primary source of prostanoids as thromboxane A2. OBJECTIVE: In this review, we summarize studies targeting prostanoids production and down-stream pathways in cardiovascular diseases, and more specifically in AAA. RESULTS AND CONCLUSION: Various inhibitors of COX or antagonists of prostanoids receptors have been investigated in AAA animal models with conflicting results. In human AAA, only a few number of studies focused on anti-platelet therapy mostly using acetylsalicylic acid (aspirin, ASA), a COX1 inhibitor. Finally, we report preliminary promising results of a model of AAA in rats receiving a thromboxane A2 inhibitor, BM-573 that induced a reduction of aneurysmal growth.

Azacytidine prevents experimental xenogeneic graft-versus-host disease without abrogating graft-versus-leukemia effects.

The demethylating agent 5-azacytidine (AZA) has proven its efficacy in the treatment of myelodysplastic syndrome and acute myeloid leukemia. In addition, AZA can demethylate FOXP3 intron 1 (FOXP3i1) leading to the generation of regulatory T cells (Treg). Here, we investigated the impact of AZA on xenogeneic graft-vs.-host disease (xGVHD) and graft-vs.-leukemia effects in a humanized murine model of transplantation (human PBMCs-infused NSG mice), and described the impact of the drug on human T cells in vivo. We observed that AZA improved both survival and xGVHD scores. Further, AZA significantly decreased human T-cell proliferation as well as IFNγ and TNF-α serum levels, and reduced the expression of GRANZYME B and PERFORIN 1 by cytotoxic T cells. In addition, AZA significantly increased Treg frequency through hypomethylation of FOXP3i1 as well as increased Treg proliferation. The latter was subsequent to higher STAT5 signaling in Treg from AZA-treated mice, which resulted from higher IL-2 secretion by conventional T cells from AZA-treated mice itself secondary to demethylation of the IL-2 gene promoter by AZA. Importantly, Tregs harvested from AZA-treated mice were suppressive and stable over time since they persisted at high frequency in secondary transplant experiments. Finally, graft-vs.-leukemia effects (assessed by growth inhibition of THP-1 cells, transfected to express the luciferase gene) were not abrogated by AZA. In summary, our data demonstrate that AZA prevents xGVHD without abrogating graft-vs.-leukemia effects. These findings could serve as basis for further studies of GVHD prevention by AZA in acute myeloid leukemia patients offered an allogeneic transplantation.

Eccentric Training for Tendon Healing After Acute Lesion: A Rat Model.

BACKGROUND: The tendon is a dynamic entity that remodels permanently. Platelet-rich plasma (PRP) injection has been shown to have a beneficial effect on tendon healing after lesion in rats. Furthermore, eccentric exercise seems to improve the mechanical quality of the tendon. HYPOTHESIS: A combination of PRP injection and eccentric training might be more effective than either treatment alone. STUDY DESIGN: Controlled laboratory study. METHODS: Adult male rats were anesthetized, an incision was performed in the middle of their left patellar tendon and an injection of physiological fluid (PF) or homologous PRP was randomly made at the lesion level. The rats were then divided into 2 groups: the eccentric group, undergoing eccentric training 3 times a week, and the untrained group, without any training. Thus, 4 groups were compared. After 5 weeks, the tendons were removed and their ultimate tensile strength and energy were measured. Tendons were frozen for proteomic analyses when all biomechanical tests were completed. Statistical analysis was performed with linear mixed effect models. RESULTS: No significant difference was found between the treatments using PF injection or PRP injection alone. However, the value of the ultimate tensile force at rupture was increased by 4.5 N (108% of control, P = .006) when eccentric training was performed. An intragroup analysis revealed that eccentric training significantly improved the ultimate force values for the PRP group. Proteomic analysis revealed that eccentric training led to an increase in abundance of several cytoskeletal proteins in the PF group, while a decrease in abundance of enzymes of the glycolytic pathway occurred in the PRP-treated groups, indicating that this treatment might redirect the exercise-driven metabolic plasticity of the tendon. CONCLUSION: Eccentric training altered the metabolic plasticity of tendon and led to an improvement of injured tendon resistance regardless of the treatment injected (PF or PRP). CLINICAL RELEVANCE: This study demonstrates the necessity of eccentric rehabilitation and training in cases of tendon lesion regardless of the treatment carried out.

Accurate Control of 17ß-Estradiol Long-Term Release Increases Reliability and Reproducibility of Preclinical Animal Studies.

Estrogens are the subject of intensive researches aiming to elucidate their mechanism of action on the various tissues they target and especially on mammary gland and breast cancer. The use of ready-to-use slow releasing devices to administer steroids, especially estrogens, to small experimental animals remains the method of choice in terms of animal well-being and of safety for both the researcher and the animal. In this study, we evaluated and compared, in vitro and in vivo, the release kinetic of estradiol (E2) over sixty days from two different slow-releasing systems: the matrix pellet (MP) and the reservoir implant (RI). We compared the impact of these systems in three E2-sensitive mouse models : mammary gland development, human MCF7 adenocarcinoma xenograft and mouse melanoma progression. The real amount of E2 that is released from both types of devices could differ from manufacturer specifications due to inadequate release for MP and initial burst effect for RI. Compared to MP, the interindividual variability was reduced with RI thanks to a superior control of the E2 release. Depending on the dose-dependent sensitivity of the physiological or pathological readout studied, this could lead to an improvement of the statistical power of in vivo experiments and thus to a reduction of the required animal number. Altogether, our data draw attention on the importance to adequately select the slow-releasing device that is the most appropriated to a specific experiment to better fulfill the 3Rs rule (Replacement, Reduction, Refinement) related to animal welfare and protection.

Impact of biomaterial microtopography on bone regeneration: comparison of three hydroxyapatites.

AIMS: The primary objective of this study was to compare the in vivo performance, namely in terms of quantity of newly formed bone and bone-to-material contact (osteoconductivity), of three hydroxyapatite-based biomaterials (HA) of different origins (natural or synthetic) or manufacturing process in a sinus lift model in rabbits. The secondary objective was to correlate the findings with the physical and topographical characteristics of the biomaterials. MATERIALS AND METHODS: Two bovine HA manufactured with different processes (bovine hydroxyapatites [BHA] and cuttlebone hydroxyapatite [CBHA]) and a synthetic hydroxyapatite (SHA) sintered at high temperature were characterised with scanning electronic microscopy (SEM) and the measurement of specific surface area (BET). The materials were implanted in a sinus lift model in rabbits; histological and histomorphometric evaluation using non-decalcified sections was performed at 1, 5 and 12 weeks after implantation. RESULTS: The studied biomaterials displayed a different surface topography. The two natural HA displayed significantly higher bone quantities (P = 0.0017; BHA vs. SHA, P = 0.0018 and CBHA vs. SHA, P = 0.033) at 5 and 12 weeks compared to the synthetic one (SHA). Moreover, the osteoconductivity (bone-to-material contact) was significantly higher in the BHA group compared to the two other groups (P = 0.014; BHA vs. SHA, P = 0.023 and BHA vs. CBHA, P = 0.033). CONCLUSION: HA-based biomaterials from diverse origins and manufacturing processes displayed different topographical characteristics. This may have influenced different regenerated bone architecture observed; more bone was found with natural HA compared to the synthetic one, and significantly higher bone-to-material contacts were found with BHA.

Limited Impact of Imatinib in a Murine Model of Sclerodermatous Chronic Graft-versus-Host Disease.

BACKGROUND: Sclerodermatous chronic Graft-versus-Host Disease (scl-cGVHD) is one of the most severe form of cGVHD. The Platelet-derived Grotwth Factor (PDGF) and the Transforming Growth Factor-ß (TGF-ß) play a significant role in the fibrosing process occurring in scl-cGVHD. This prompted us to assess the impact of the PDGF-r and c-Abl tyrosine kinase inhibitor imatinib on scl-cGVHD. METHODS: To assess the impact of imatinib on T cell subset proliferation in vivo, Balb/cJ recipient mice were lethally (7 Gy) irradiated and then injected with 10x106 bone marrow cells from B10.D2 mice on day 0. Fourteen days later, 70x106 carboxyfluorescein succinimidyl ester (CFSE)-labeled splenocytes from B10.D2 mice were infused and imatinib or sterile water was administered for 5 days. To induce severe scl-cGVHD, Balb/cJ mice were injected i.v. with 10.106 bone marrow cells and 70.106 splenocytes from B10.D2 donor mice after 7 Gy irradiation. Mice were then given sterile water or imatinib from day +7 after transplantation to the end of the experiment (day +52). RESULTS: Imatinib decreased the proliferation of total T cells (P = 0.02), CD8+ T cells (P = 0.01), and of regulatory T cells (Tregs) (P = 0.02) in the spleen. In the severe scl-cGVHD model, imatinib-treated mice had significantly lower levels of PDGF-r phosphorylation than control mice on day 29 after transplantation (P = 0.008). However, scl-cGVHD scores were similar between vehicle- and imatinib-treated mice during the whole experiment, while there was a suggestion for less weight loss in imatinib-treated mice that reached statistical significance at day +52 following transplantation (P = 0.02). CONCLUSIONS: Imatinib had a limited impact in murine scl-cGVHD despite significant inhibition of PDGF-r.

68Ga and 188Re Starch-Based Microparticles as Theranostic Tool for the Hepatocellular Carcinoma: Radiolabeling and Preliminary In Vivo Rat Studies.

PURPOSE: This work aims to develop, validate and optimize the radiolabeling of Starch-Based Microparticles (SBMP) by 188Re and 68Ga in the form of ready-to-use radiolabeling kits, the ultimate goal being to obtain a unique theranostic vector for the treatment of Hepatocellular Carcinoma. METHODS: Optimal labeling conditions and composition of freeze-dried kits were defined by monitoring the radiochemical purity while varying several parameters. In vitro stability studies were carried out, as well as an in vivo biodistribution as a preliminary approach with the intra-arterial injection of 68Ga radiolabeled SBMP into the hepatic artery of DENA-induced rats followed by PET/CT imaging. RESULTS: Kits were optimized for 188Re and 68Ga with high and stable radiochemical purity (>95% and >98% respectively). The in vivo preliminary study was successful with more than 95% of activity found in the liver and mostly in the tumorous part. CONCLUSION: SBMP are a promising theranostic agent for the Selective Internal Radiation Therapy of Hepatocellular carcinoma.

Sepramesh and postoperative peritoneal adhesions in a rat model.

PURPOSE: The present study aimed to investigate the safety and the anti-postoperative peritoneal adhesion (PPA) characteristics of Sepramesh® (Davol), a composite mesh made of polypropylene covered with Seprafilm, when intraperitoneally placed in a rat model. METHODS: Twenty male rats were randomized into a control group and a Sepramesh group. They underwent a primary surgical procedure aiming to induce a peritoneal injury in order to induce PPAs. In the Sepramesh group, the burnt peritoneum was covered with a 2-cm diameter disc of Sepramesh prosthesis. The mesh was fixed to the parietal peritoneum with four 3-0 absorbable stitches. PPAs were assessed during a second laparotomy 10 days later using quantitative and qualitative scoring systems. RESULTS: There was no difference in terms of mean number of PPAs between both groups. All the rats from the control group developed PPAs. In the Sepramesh group, no adhesions were observed at the site of the injured peritoneum that had been covered with the Sepramesh prosthesis, but PPAs occurred at the extremities of the mesh, where there was close contact between polypropylene and viscera, or where the fixation sutures were placed. The severity and the type of adhesions were significantly higher in the control group. CONCLUSIONS: This study demonstrated that for the Sepramesh prostheses, the Seprafilm layer might be effective in PPA prevention, but damage caused by the section and fixation of Sepramesh should be limited in order to limit PPAs.

Azacytidine mitigates experimental sclerodermic chronic graft-versus-host disease.

BACKGROUND: Previous studies have demonstrated that regulatory T cells (Tregs) play a protective role in the pathogenesis of chronic graft-versus-host disease (cGVHD). Tregs constitutively express the gene of the transcription factor Foxp3 whose CNS2 region is heavily methylated in conventional CD4(+) T cells (CD4(+)Tconvs) but demethylated in Tregs. METHODS: Here, we assessed the impact of azacytidine (AZA) on cGVHD in a well-established murine model of sclerodermic cGVHD (B10.D2 (H-2d) → BALB/cJ (H-2d)). RESULTS: The administration of AZA every 48 h from day +10 to day +30 at the dose of 0.5 mg/kg or 2 mg/kg mitigated chronic GVHD. Further, AZA-treated mice exhibited higher blood and thymic Treg frequencies on day +35, as well as higher demethylation levels of the Foxp3 enhancer and the IL-2 promoter in splenocytes at day +52. Interestingly, Tregs from AZA-treated mice expressed more frequently the activation marker CD103 on day +52. AZA-treated mice had also lower counts of CD4(+)Tconvs and CD8(+) T cells from day +21 to day +35 after transplantation, as well as a lower proportion of CD4(+)Tconvs expressing the Ki67 antigen on day +21 demonstrating an anti-proliferating effect of the drug on T cells. CONCLUSIONS: Our results indicate that AZA prevented sclerodermic cGVHD in a well-established murine model of cGVHD. These data might serve as the basis for a pilot study of AZA administration for cGVHD prevention in patients at high risk for cGVHD.

Multimodality imaging assessment of the deleterious role of the intraluminal thrombus on the growth of abdominal aortic aneurysm in a rat model.

OBJECTIVES: To evaluate imaging changes occurring in a rat model of elastase-induced abdominal aortic aneurysm (AAA), with emphasis on the intraluminal thrombus (ILT) occurrence. METHODS: The post-induction growth of the AAA diameter was characterized using ultrasound in 22 rats. ILT was reported on 13 rats that underwent 14 magnetic resonance imaging (MRI) 2-18 days post-surgery, and on 10 rats that underwent 18 fluoro-deoxyglucose (FDG) positron emission tomography (PET)/microcomputed tomography examinations 2-27 days post-surgery. Logistic regressions were used to establish the evolution with time of AAA length, diameter, ILT thickness, volume, stratification, MRI and FDG PET signalling properties, and histological assessment of inflammatory infiltrates. RESULTS: All of the following significantly increased with time post-induction (p < 0.001): AAA length, AAA diameter, ILT maximal thickness, ILT volume, ILT iron content and related MRI signalling changes, quantitative uptake on FDG PET, and the magnitude of inflammatory infiltrates on histology. However, the aneurysm growth peak followed occurrence of ILT approximately 6 days after elastase infusion. CONCLUSION: Our model emphasizes that occurrence of ILT precedes AAA peak growth. Aneurysm growth is associated with increasing levels of iron, signalling properties changes in both MRI and FDG PET, relating to its biological activities. KEY POINTS: • ILT occurrence in AAA is associated with increasing FDG uptake and growth. • MRI signalling changes in ILT reflect activities such as haemorrhage and RBC trapping. • Monitoring ILT activities using MRI may require no exogenous contrast agent.

Asporin Is a Fibroblast-Derived TGF-ß1 Inhibitor and a Tumor Suppressor Associated with Good Prognosis in Breast Cancer.

BACKGROUND: Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-ß1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications. METHODS AND FINDINGS: Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1ß, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-ß1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold; p = 0.001). Assessment of asporin expression and patient outcome (n = 60; 10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87; 95% CI 0.78-0.96; p = 0.0001). Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37-0.91; p = 0.017). Although these data highlight the potential of asporin to serve as a prognostic marker, confirmation of the clinical value would require a prospective study on a much larger patient cohort. CONCLUSIONS: Our data show that asporin is a stroma-derived inhibitor of TGF-ß1 and a tumor suppressor in breast cancer. High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome. Future pre-clinical studies should consider options for increasing asporin expression in TNBC as a promising strategy for targeted therapy.

Preovulatory follicle diameter, growth rate and time of ovulation during induced oestrus using a CIDR® in trypanotolerant female Bos taurus N'Dama cattle.

The aim of this study was to assess the dose (300 to 600 IU) effects of equine chorionic gonadotropin (eCG) on the preovulatory follicle diameter, growth rate and time of ovulation characterized by echography. The eCG was injected at the end (D0) of the 7-day treatment with a controlled internal device release (CIDR®) and a PGF2α being injected 2 days before the removal of the CIDR® (d-2). The 120 N'Dama female were distributed into five experimental groups. The control group (n = 26) was treated with physiological saline at the removal of the CIDR®, while the animals in the four treated groups received, respectively, 300 IU (n = 25), 400 IU (n = 24), 500 IU (n = 22) and 600 IU (n = 23) of eCG. The diameter of the preovulatory follicle was significantly higher (P < 0.05) in the animals treated with 300 IU (10.1 ± 1.4 mm) than in untreated animals (9.3 ± 1.2 mm). Follicle growth rate was significantly (P < 0.05) higher in treated animals (1.0 ± 0.4 mm/day) than in the control group (0.9 ± 0.4 mm/day). The average interval between the time of eCG injection and ovulation was similar in the non-treated (83.7 ± 14.4 h) and treated animals (79.7 ± 11.9). Treated animals showed a significant increase in the percentage of ovulation (94.7 % compared to 73.1 %) (P < 0.01). Use of eCG contributed towards synchronising the time of ovulation between 72 to 96 h, which would facilitate the use of systematic insemination.