4‐Thio‐deoxyuridylate‐modified thrombin aptamer and its inhibitory effect on fibrin clot formation,platelet aggregation and thrombus growth on subendothelial matrix

Summary. Background: The consensus thrombin aptamer C15‐mer is a single‐stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin‐binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4‐thio‐deoxyuridylate (s4dU)‐containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin‐inhibiting sequence, in which some of the thymidylate residues were replaced by 4‐thio‐deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin‐catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin‐induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin‐treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15‐mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15‐mer) showed a 2‐fold increased inhibition of thrombin‐catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin‐treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin‐induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15‐mer was 3‐fold and twelve‐fold more effective than C15‐mer, respectively. Conclusion: The replacement of four thymidylate residues in C15‐mer by 4‐thio‐deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.     

人尿中脱氢睾酮代谢物的GC/MS分析

本文用GC/MS方法,对人尿中脱氢睾酮(boldenone)代谢物进行了研究。志愿者口服20 mg脱氢睾酮后,收集阳性尿。尿样经过包括XAD-2树脂柱吸附、酶水解、有机相萃取及三甲基硅烷衍生化反应的预处理后,用毛细管气相色谱与质谱检测器联用进行分析,鉴定了脱氢睾酮的几个主要代谢物和它们的代谢模式;总结了兴奋剂检测中具有意义的代谢物及其碎片离子;对收集的阳性尿中脱氢睾酮浓度进行测定;对预处理方法的回收率进行了研究。     

Aorto-enteric fistula development secondary to mycotic abdominal aortic aneurysm following intravesical bacillus Calmette–Guerin (BCG) treatment for transitional cell carcinoma of the bladder

INTRODUCTIONIntravesical BCG-instillation for bladder cancer is considered safe but is not without risk. While most side-effects are localised and self-limiting, the development of secondary vascular pathology is a rare but significant complication.PRESENTATION OF CASEA 77-year-old male presented with a mycotic abdominal aortic aneurysm and associated aorto-enteric fistula 18 months after receiving intravesical BCG-instillations for early stage transitional cell carcinoma.DISCUSSIONResponse rates to intravesical BCG for early stage transitional cell carcinoma are high. The procedure produces a localised inflammatory response in the bladder but the exact mechanism of action is unclear. The treatment is generally well tolerated but BCG-sepsis and secondary vascular complications have been documented.Mycotic abdominal aortic aneurysm with associated aorto-enteric fistula secondary to BCG is very rare. Few examples have been documented internationally and the extent of corresponding research and associated management proposals is limited.Surgical options include in situ repair with prosthetic graft, debridement with extra-anatomical bypass and, occasionally, endovascular stent grafting. Recommended medical therapy for systemic BCG infection is Isoniazid, Rifampicin and Ethambutol.CONCLUSIONCurrent screening methods must be updated with clarification regarding duration of anti-tuberculous therapy and impact of concomitant anti-tuberculous medication on the therapeutic action of intravesical BCG. Long-term outcomes for patients post graft repair for mycotic aneurysm are unknown and more research is required regarding the susceptibility of vascular grafts to mycobacterial infection.Recognition of the risks associated with BCG-instillations, even in immunocompetent subjects, is paramount and must be considered even several months or years after receiving the therapy.     

Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524–543 reduces cyclophosphamide-accelerated diabetes

NOD mice constitute a model for studying the prevention of human autoimmune type 1 diabetes. Glutamic acid decarboxylase (GAD) could be a key antigen involved in this disease, and GAD65 peptide 524–543 has been implicated in early T cell response in young NOD mice. We performed two i.p. injections of GAD peptide 524–543 (100 μg at each injection), together with Freund*s incomplete adjuvant (FIA), into female NOD mice at 30 and 45 days old. Diabetes was accelerated 2 weeks later by a single injection of cyclophosphamide (CY), which acts against suppressive mechanisms. Treatment with GAD 524–543 peptide delayed the onset of diabetes and reduced its incidence (28% versus 60%; P<0·001) compared with control mice injected with FIA alone, or GAD peptide 534–553, or an irrelevant peptide. In the same group, the severity of lymphocytic inflammation of pancreatic islets was reduced (P<0·03). Up to 3 months after peptide injections, a strong splenocytic proliferative response occurred in immunized NOD mice against the immunizing peptide alone (but not against a panel of seven other GAD65-derived peptides). After peptide challenge of splenocytes in vitro, protection against CY-accelerated diabetes was associated with higher peptide-specific production of T helper type 2 (Th2)-associated interleukins 4 and 10, whereas Th1-associated interferon-gamma and IL-2 were proportionally less represented. During cotransfer, T splenocytes from GAD 524–543-immunized mice were able to reduce the capacity of T cells from diabetic donors to transfer the disease adoptively (P<0·01), demonstrating the generation of cellular mechanisms that actively suppress the disease. It is concluded that immunization of NOD mice with GAD65 peptide 524–543 can counteract CY-accelerated diabetes, possibly through active cellular suppression linked to a shift of Th1/Th2 balance toward the production of Th2 cytokines such as IL-4 and IL-10. This study provides additional support for the notion that GAD, and more precisely its epitope 524–543, could be one of the key targets for the pathogenesis of type 1 diabetes in NOD mice, as well as for the efficacy of disease-specific peptide therapy in type 1 diabetes.     

Efficacy and tolerability of 8 weeks'' treatment with terbinafine in children with tinea capitis caused by Microsporum canis: a comparison of three doses

BACKGROUND: Tinea capitis caused by Microsporum canis is the most common mycosis of the scalp in preschool and school-aged children in Greece. OBJECTIVE: To compare the efficacy, safety and tolerability of an 8-week course of oral terbinafine at different doses. METHODS: Patients received oral terbinafine at doses ranging from 3.3 to 12.5 mg/kg/day for 8 weeks, as follows: group A, terbinafine 3.3 to 6.0 to 7.0 mg/kg/day (23 patients); group C, terbinafine > 7.0 to 12.5 mg/kg/day (37 patients). Fungal microscopy and cultures were performed 4 weeks before the start of the treatment, at the end of the treatment (week 8) and at a follow-up visit at week 16. RESULTS: At week 8 mycological cure was achieved in one patient (2.7%) in group A, in 21 patients (91.3%) in group B and in 34 patients (97.1%) in group C. At week 16 mycological cure was achieved in one patient (2.7%) in group A, in 22 patients (95.7%) in group B and in 35 patients (100%) in group C. There was a statistically significant difference (P < 0.0005) between dose level and efficacy of terbinafine at the end of the treatment period and also at the follow-up visit at week 16. Five patients (three in group A and two in group C) discontinued treatment because of adverse events. CONCLUSIONS: The administration of terbinafine at a dose of either 6-7 or 7-12.5 mg/kg/day for 8 weeks is safe and effective for the treatment in children of tinea capitis caused by M. canis.     

DETECTION OF PROSTATE CANCER WITH C-METHIONINE POSITRON EMISSION TOMOGRAPHY

PurposeWe studied the detection of primary prostate cancer with positron emission tomography (PET) using ¹¹C-labeled methionine (MET) in patients with increased prostate specific antigen (PSA) levels and repeatedly negative biopsies.Materials and MethodsA total of 20 consecutive patients with increased serum PSA and negative repeat biopsies were included in the study. Patient age ranged from 52 to 75 years (average 65). PSA levels ranged from 3.49 to 28.6 ng/ml (average 9.36). Dynamic PET images were obtained from the prostate region using ¹¹C-labeled MET. Suspicious accumulations of the tracer were anatomically localized using magnetic resonance images and were used as guidance during the next biopsy.ResultsPET was positive in 15 (75%) patients, in 7 of whom (46.7%) the next repeat biopsy verified carcinoma. The overall detection rate was 35% (7 of 20) and 46.7% (7 of 15) in the whole group and in the positive PET group, respectively. All 5 of 5 patients with negative MET PET had negative biopsies.ConclusionsMET PET of the prostate with short dynamic scanning and multicore biopsy is a useful method to ensure a high detection rate of prostate cancer in patients with increased PSA and repeat negative biopsies.     

Antigenic characterization of endothelial cell-derived microparticles and their detection ex vivo

Summary. Background: Endothelial activation and dysfunction are associated with several diseases. However, hardly any specific markers are available. Microparticles (MP) from endothelial cells (EC; EMP) were reported in patient groups and healthy individuals. The antibodies used to detect EMP, however, were mainly directed against antigens without EC specificity. Objectives: We evaluated the antigens on EC and EMP to establish proper markers for EMP detection. Methods: EMP were isolated from supernatants of resting and interleukin (IL)‐1α activated human umbilical vein EC (HUVEC; n = 3; 0–72 h), stained with annexin V and monoclonal antibodies, and analyzed by flow cytometry. Human platelet‐MP (PMP), the main MP population in plasma, were prepared in vitro. EMP and PMP were studied in plasma from systemic lupus erythematosus (SLE) patients (n = 11) and healthy individuals (n = 10). Results: Platelet–endothelial cell adhesion molecule‐1 (PECAM‐1), α_ν and β₃ were constitutively exposed on HUVEC, but (almost) absent on EMP (<15% positive for α_ν and β₃), or only exposed on a subpopulation (PECAM‐1; 30–60%). Activated HUVEC (>80%) and (subpopulations of) EMP exposed E‐selectin and tissue factor. PMP strongly exposed PECAM‐1, β₃, and glycoprotein (GP)Ib (CD42b), but not α_ν or E‐selectin. GPIb and P‐selectin (CD62P) were absent on EMP. Plasma samples contained 0.5% MP staining for E‐selectin and/or α_ν. Plasma from one SLE patient contained E‐selectin exposing MP (21%), but little α_ν‐positive MP. Conclusions: EC release EMP in vitro. The antigenic phenotype of EMP released from resting and IL‐1α‐stimulated EC differs among each other as well as from resting and stimulated EC, respectively. E‐selectin exposed on IL‐1α‐stimulated EC is a valid marker for EMP detection ex vivo to establish endothelial cell activation.