A novel and potent thrombolytic fusion protein consisting of anti-insoluble fibrin antibody and mutated urokinase

Tissue plasminogen activator (tPA) is used clinically because it has higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity in places other than IF. UK has the advantage that it is specifically activated on IF, but it binds IF weakly. Previously, we established a monoclonal antibody (mAb) that recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the reduction of plasma plasminogen levels in vivo as compared to UK. In the photo-chemically induced thrombus mouse model, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA, and 90% (9/10) in the AMU1114 treatment group. Although no death was observed 1 hour after administration of each thrombolytic agent, some dead mice were identified within 24 hours in all treatment groups including control. These data indicate the need for further basic studies of AMU1114.

Diagnostic and procedural intraoperative ultrasound: technique, tips and tricks for optimizing results

Intraoperative ultrasound (IOUS) is a valuable adjunctive tool that can provide real-time diagnostic information in surgery that has the potential to alter patient management and decrease complications. Lesion localization, characterization and staging can be performed, as well as surveying for additional lesions and metastatic disease. IOUS is commonly used in the liver for hepatic metastatic disease and hepatocellular carcinoma, in the pancreas for neuroendocrine tumors, and in the kidney for renal cell carcinoma. IOUS allows real-time evaluation of vascular patency and perfusion in organ transplantation and allows for early intervention for anastomotic complications. It can also be used to guide intraoperative procedures such as biopsy, fiducial placement, radiation, or ablation. A variety of adjuncts including microbubble contrast and elastography may provide additional information at IOUS. It is important for the radiologist to be familiar with the available equipment, common clinical indications, technique, relevant anatomy and intraoperative imaging appearance to optimize performance of this valuable imaging modality.

Modeling changes in vascular and extracellular matrix integrity that can occur in decellularized kidneys after implantation

A method was established to identify alterations in vascular patency and extracellular matrix integrity of decellularized porcine kidney scaffolds. These scaffolds were perfused with blood at physiologically normal (500 and 650 ml/min) and abnormal (200 ml/min) rates. Variations in venous outflow were then assessed over 24 hours. Angiographic data confirmed that standard arterial branching patterns and the integrity of the extracellular matrix were considerably disrupted. Scaffolds subjected to normal arterial perfusion rates observed drops in venous outflow across the 24 hours. These reductions rose from roughly 40% after 12 hours to 60% after 24 hours. At the end of the test period, regardless of the underlying damage that occurred, the kidneys appeared intact on the surface, and there were no apparent signs of clotting. In comparison, venous flow rates decreased by 80 to 100% across the 24 hours in acellular scaffolds subjected to a far lower perfusion rate of 200 ml/min. These kidneys also appeared intact after 24 hours of perfusion, but presented several arterial, venous, and ureteral clots. The results of this study provide insight into circumstances that limit scaffold viability and provide a simplified model to analyze other conditions that can better prepare scaffolds for long-term transplantation.

A novel and potent thrombolytic fusion protein consisting of anti-insoluble fibrin antibody and mutated urokinase

Because the risk of thromboembolism increases with age, as well as due to infectious diseases, safer and more effective thrombolytic agents are in greater demand. Tissue plasminogen activator (tPA) is currently used clinically because it has higher binding specificity for insoluble fibrin (IF) than urokinase (UK), but even pro-tPA has catalytic activity in places other than IF. Meanwhile, UK has the advantage that it is specifically activated on IF, but it only binds IF weakly. Unlike the anti-IF monoclonal antibody (mAb) established in the past, our anti-IF mAb recognizes a pit structure formed only in IF. Here, we developed a new mAb against the pit, 1101, that does not affect coagulation or fibrinolysis, and prepared a fusion protein of UK with humanized 1101 Fab to transport UK selectively to IF. In IF-containing lesions, UK is cleaved by plasmin at two sites, Lys158/Ile159 and Lys135/Lys136. Cleavage of the former leads to activation of UK; however, because activated UK is linked by S-S bonds before and after cleavage, it is not released from the fusion. Cleavage at the latter site causes UK to leave the fusion protein; hence, we mutated Lys135/Lys136 to Gly135/Gly136 to prevent release of UK. This engineered UK-antibody fusion, AMU1114, significantly decreased the systemic side effects of UK in vivo. In a mouse thrombus formation experiment, the vascular patency rate was 0% (0/10) in the control, 50% (5/10) in the tPA, and 90% (9/10) in the AMU1114 treatment group. These data support future clinical development of AMU1114.

Influence of components of optical momentum and spin of evanescent waves on micro- and nanoobjects (Review)

Background: Mechanical properties of light are widely used in applied areas, such as optical trapping and manipulation, sorting, deformation of biological cells and molecules. In general, the evanescent field may exhibit three components of optical momentum and spin angular momentum (spin), which manifest themselves in the occurrence of corresponding components of optical force and torque. Such extraordinary properties of evanescent waves open up new possibilities for manipulating of micro- and nanoobjects, in comparing with classical optical tweezers and manipulators, which can be used for solving the applied problems, in particular, of biomedicine. Objectives: Aim of this work is to analyze and summarize recent studies regarding to the mechanical influence of evanescent field on micro- and nanoobjects, in particular, related to the influence of transverse components of optical momentum and spin. Materials and methods: Method of momenta allows one to distinguish in an evanescent field the action of optical forces and torques, associated with the components of optical momentum and angular momentum of different nature and action direction, depending on the polarization of the incident wave. Experimental methods of particle manipulation in the near field allow visualizing such an influence, which makes it possible for solving the applied problems. Results: Recent studies demonstrate the action on nano- and microobjects of such "extraordinary" optical momentum and spin components, as transverse spin momentum, transverse spin, transverse imaginary optical momentum component, and vertical spin. Using, in particular, the latter, to solve the applied problems of biomedicine is proposed, such as transporting of therapeutic agents to pathological areas or restoring vascular patency and tissue blood supply. Conclusions: Obtained results of theoretical and experimental investigation of the mechanical action of the optical momentum and spin components of evanescent field allow us to extend the approaches of optical manipulation of micro- and nanoobjects, with the possibility of applications, in particular, for the problems of biomedicine.

Clinical and radiological follow-up after free vascularised fibula transplantation for bone defects of the upper extremity with MRI and MR-angiography

Background Vascular patency of free vascularised fibula grafts can be postoperatively assessed by island flap which is not always possible or angiography which is invasive. Bone healing is examined based on radiographs with scoring systems. We present data on MRI with contrast agent and a comparison of the Giessler bone healing score by X-ray and MRI and clinical scores in patients with operation on the upper extremity. Methods We reviewed the clinical and radiological outcome of 13 patients with a follow-up of at least 1 year after free vascularised fibula graft of the upper extremity. The examination included the DASH, MSTS, SF-36, Rosén-Lundberg-scores and MRI with contrast agent. We determined the Giessler bone healing score by most recent x-ray and MRI and the Weiland score and its modification for the upper extremity. For statistics, we used the Wilcoxon test and Pearson correlation. Results Vascular patency was directly or indirectly detectable all cases in the MRI with contrast agent. 5 patients did not return to work and 5 patients had ongoing pain medication. Median DASH was 21.7, MSTS of the upper extremity 76.7, of the lower extremity 93.3, the Rosén-Lundberg score for median nerve 2.43, for ulnar nerve 2.32. Giessler score for x-ray and MRI showed no significant difference for proximal or distal junction. Conclusions We could prove with MRI that it was indeed a vascularised graft and its vitality at time of follow-up by assessment of vascular patency, and that MRI can be used to determine the Giessler score similarly to X-ray.