Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.     

Loading deproteinized bovine bone with strontium enhances bone regeneration in rat calvarial critical size defects

Clinical Oral Investigations - To evaluate the effect of grafting with strontium (Sr)-loaded deproteinized bovine bone (DBB) on bone healing in calvarial critical size defects (CSD) in rats. Two...     

Small and similar amounts of micromotion in an anatomical stem and a customized cementless femoral stem in regular-shaped femurs: A 5-year follow-up randomized RSA study

Background and purpose

High primary stability is important for long-term survival of uncemented femoral stems. Different stem designs are currently in use. The ABG-I is a well-documented anatomical stem with a press-fit design. The Unique stem is designed for a tight customized fit to the cortical bone of the upper femur. This implant was initially developed for patients with abnormal anatomy, but the concept can also be used in patients with normal femoral anatomy. We present 5-year radiostereometric analysis (RSA) results from a randomized study comparing the ABG-I anatomical stem with the Unique femoral stem.

Patients and methods

100 hips with regular upper femur anatomy were randomized to either the ABG-I stem or the Unique femoral stem. RSA measurements were performed postoperatively and after 3, 6, 12, 24, and 60 months.

Results

RSA measurements from 80 hips were available for analysis at the 5-year follow-up. Small amounts of movement were observed for both stems, with no statistically significant differences between the 2 types.

Interpretation

No improvement in long-term stability was found from using a customized stem design. However, no patients with abnormal geometry of the upper femur were included in this study.High mechanical stability is a crucial factor for correct performance of uncemented femoral stems. Micromovements along the implant-bone interface may prevent ingrowth of bone to the surface of the prosthesis, and it may lead to the formation of a fibrous membrane and eventually to loosening of the implant. The critical thresholds of micromovements that can be tolerated are not exactly known, but they are probably dependent on both patient- and implant-specific factors (Viceconti et al. 2006). It has been shown, however, that interfacial motion of around 40 μm leads to partial bone ingrowth whereas motions exceeding 150 μm completely prevent ingrowth of bone (Pilliar et al. 1986, Jasty et al. 1997).Uncemented, customized femoral stems are mainly designed and manufactured for patients with abnormal size and shape of the proximal femur, but this does not preclude their use in patients with regular-shaped proximal femurs. The requirement for maximum primary stability with uncemented off-the-shelf stems also applies to customized stems. The optimized fit and fill of a customized stem should theoretically promote even better mechanical fixation than with standard implants.Radiostereometric analysis (RSA) enables measurement of migration and rotation in the range of 0.1 mm and 0.05º, respectively (Selvik 1989, Kadar et al. 2011). There is a correlation between postoperative migration of femoral stems and early loosening (Freeman and Plante-Bordeneuve 1994, Linder 1994, Karrholm et al. 2006, Karrholm 2012). On the other hand, a new implant showing large degrees of micromovement should not necessarily be regarded as having a performance equivalent to long-term failure (Karrholm et al. 2006). Recently published studies reporting medium- to long-term RSA results will probably contribute to a better understanding of the topic (Nieuwenhuijse et al. 2012, Rohrl et al. 2012).This randomized study was performed as part of the clinical documentation of the Unique customized stem (Scandinavian Customized Prosthesis (SCP), Trondheim, Norway), to compare the migration pattern of the Unique stem with that of a standard anatomical uncemented stem with a clinically well-proven stem design (the ABG-I).Our aim was to measure migration of the Unique customized stem and the ABG-I stem using RSA. Our hypothesis was that there would be no difference in migration between the 2 types of uncemented femoral stems in patients with regular anatomy in the upper femur.     

Pelvic irradiation does not increase the risk of hip replacement in patients with gynecological cancer: A cohort study based on 8,507 patients

Background and purpose —

Long-term survivors of cancer can develop adverse effects of the treatment. 60% of cancer patients survive for at least 5 years after diagnosis. Pelvic irradiation can cause bone damage in these long-term survivors, with increased risk of fracture and degeneration of the hip.

Patients and methods —

Analyses were based on linkage between the Cancer Registry of Norway (CRN) and the Norwegian Arthroplasty Register (NAR). All women who had been exposed to radiation for curative radiotherapy of gynecological cancer (40–60 Gy for at least 28 days) were identified in the CRN. Radiotherapy had been given between 1998 and 2006 and only patients who were irradiated within 6 months of diagnosis were included. The control group contained women with breast cancer who had also undergone radiotherapy, but not to the pelvic area. Fine and Gray competing-risk analysis was used to calculate subhazard-rate ratios (subHRRs) and cumulative incidence functions (CIFs) for the risk of having a prosthesis accounting for differences in mortality.

Results —

Of 962 eligible patients with gynecological cancer, 26 (3%) had received a total hip replacement. In the control group without exposure, 253 (3%) of 7,545 patients with breast cancer had undergone total hip replacement. The 8-year CIF for receiving a total hip replacement was 2.7% (95% CI: 2.6–2.8) for gynecological cancer patients and 3.0% (95% CI: 2.95–3.03) for breast cancer patients; subHRR was 0.80 (95% CI: 0.53–1.22; p = 0.3). In both groups, the most common reason for hip replacement was idiopathic osteoarthritis.

Interpretation —

We did not find any statistically significantly higher risk of undergoing total hip replacement in patients with gynecological cancer who had had pelvic radiotherapy than in women with breast cancer who had not had pelvic radiotherapy.After approximately 5 years, two-thirds of cancer patients are still alive (Sant et al. 2009). Research on late adverse effects in cancer survivors has gained increasing interest over the last decade. However, the main interest has been on secondary cancer events (Curtis et al. 2006), cardiovascular complications, and emotional problems (Meyerowitz et al. 2008). The relationship between cancer, skeletal disorders, and treatment has rarely been investigated. Skeletal adverse effects of irradiation include cell death, cellular injury, and abnormal bone repair—although the underlying mechanisms are not fully understood (Yurut-Caloglu et al. 2010).Pelvic insufficiency fractures may be one of the possible late side effects of radiotherapy to the pelvic area. The incidence of such fractures has been reported to be 5–20% in gynecological cancer patients who have undergone radiotherapy (Kwon et al. 2008, Oh et al. 2008, Schmeler et al. 2010, Shih et al. 2013). Less common effects of pelvic irradiation include acetabular protrusion and avascular necrosis of the femoral head (Fiorino et al. 2009). Other studies have not found any increased risk of hip fracture after pelvic irradiation (Feltl et al. 2006, Elliott et al. 2011). We wanted to examine the risk of receiving a total hip replacement in patients with cancer in the pelvic area who had undergone radiotherapy compared to patients with cancer at another location who had not undergone pelvic irradiation. Our hypothesis was that patients who have had high-dose pelvic radiotherapy would have a higher risk of undergoing total hip replacement than women who have had radiotherapy with target fields in other parts of the body.     

The impact of early cytomegalovirus infection after kidney transplantation on long‐term graft and patient survival

This prospective observational cohort study is an extension of a previous study reporting effects of cytomegalovirus (CMV) on graft and patient survival in 471 patients who underwent kidney transplantation between 1994 and 1997. CMV pp65 antigen was measured every 7–14 d during the first three months after transplantation, given as number of CMV pp65‐positive cells per 10⁵ leukocytes. A positive test was defined as CMV infection. None of the patients received CMV prophylaxis or preemptive treatment. During a median of 13.7 (7.1–14.9) yr, the number of death‐censored graft losses was 118 (25%) and of patient deaths 224 (48%). CMV infection was an independent significant risk factor for mortality in multivariate analysis (HR = 1.453, 95% CI 1.033–2.045, p = 0.032), adjusting for patient and donor age, preemptive transplantation, HLA‐DR and ‐AB mismatches, living donor, acute rejection during the first three months, donor–recipient CMV IgG antibody status and diabetic nephropathy. In univariate analysis, CMV infection was significantly associated with death‐censored graft loss but the association was not significant in multivariate model. CMV infection early after kidney transplantation is a predictor of overall mortality but not of death‐censored graft loss after a median observation period of 13.7 yr.     

Real-time biofeedback integrated into neuromuscular training reduces high-risk knee biomechanics and increases functional brain connectivity: A preliminary longitudinal investigation

Prospective evidence indicates that functional biomechanics and brain connectivity may predispose an athlete to an anterior cruciate ligament injury, revealing novel neural linkages for targeted neuromuscular training interventions. The purpose of this study was to determine the efficacy of a real-time biofeedback system for altering knee biomechanics and brain functional connectivity. Seventeen healthy, young, physically active female athletes completed 6 weeks of augmented neuromuscular training (aNMT) utilizing real-time, interactive visual biofeedback and 13 served as untrained controls. A drop vertical jump and resting state functional magnetic resonance imaging were separately completed at pre- and posttest time points to assess sensorimotor adaptation. The aNMT group had a significant reduction in peak knee abduction moment (pKAM) compared to controls (p = .03, d = 0.71). The aNMT group also exhibited a significant increase in functional connectivity between the right supplementary motor area and the left thalamus (p = .0473 after false discovery rate correction). Greater percent change in pKAM was also related to increased connectivity between the right cerebellum and right thalamus for the aNMT group (p = .0292 after false discovery rate correction, r² = .62). No significant changes were observed for the controls (ps > .05). Our data provide preliminary evidence of potential neural mechanisms for aNMT-induced motor adaptations that reduce injury risk. Future research is warranted to understand the role of neuromuscular training alone and how each component of aNMT influences biomechanics and functional connectivity.     

High-dose rifampin improves bactericidal activity without increased intracerebral inflammation in animal models of tuberculous meningitis

Tuberculous meningitis (TB meningitis) is the most severe form of tuberculosis (TB), requiring 12 months of multidrug treatment for cure, and is associated with high morbidity and mortality. High-dose rifampin (35 mg/kg/d) is safe and improves the bactericidal activity of the standard-dose (10 mg/kg/d) rifampin-containing TB regimen in pulmonary TB. However, there are conflicting clinical data regarding its benefit for TB meningitis, where outcomes may also be associated with intracerebral inflammation. We conducted cross-species studies in mice and rabbits, demonstrating that an intensified high-dose rifampin-containing regimen has significantly improved bactericidal activity for TB meningitis over the first-line, standard-dose rifampin regimen, without an increase in intracerebral inflammation. Positron emission tomography in live animals demonstrated spatially compartmentalized, lesion-specific pathology, with postmortem analyses showing discordant brain tissue and cerebrospinal fluid rifampin levels and inflammatory markers. Longitudinal multimodal imaging in the same cohort of animals during TB treatment as well as imaging studies in two cohorts of TB patients demonstrated that spatiotemporal changes in localized blood-brain barrier disruption in TB meningitis are an important driver of rifampin brain exposure. These data provide unique insights into the mechanisms underlying high-dose rifampin in TB meningitis with important implications for developing new antibiotic treatments for infections.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.