Viability of Human Arterial Grafts Monitored by Comet Assay.

An analytical method for studying DNA degradation by electrophoresis after cell lysis and visualization of DNA fragments with fluorescent dye, comet assay, was used to evaluate the viability of the endothelial layer of human arterial grafts with the aim of identifying the procedure that will least damage the tissue before cryopreservation. Four groups of samples were studied: cryopreserved arterial grafts that were thawed in two different ways, slowly lasting 2 hours or rapidly for approx. 7 minutes. Arterial grafts that were collected as part of multiorgan procurement with minimal warm ischemia time. Cadaveric grafts were taken as part of the autopsy, so they have a more extended period of warm ischemia. The HeadDNA (%) parameter and others commonly used parameters like TailDNA (%). TailMoment, TailLength, OliveMoment, TailMoment to characterize the comet were used to assess viability in this study. The ratio of non-decayed to decayed nuclei was determined from the values found. This ratio for cadaveric grafts was 0.63, for slowly thawed cryopreserved grafts 2.9, for rapidly thawed cryopreserved grafts 1.9, and for multi-organ procurement grafts 0.68. The results of the study confirmed the assumption that the allografts obtained from cadaveric donors are the least suitable. On the other hand, grafts obtained from multiorgan donors are better in terms of viability monitored by comet assay. Keywords: Arterial grafts, Cryopreservation, Cadaveric, Multiorgan procurement, Viability, Comet assay.

[The EDEC curriculum-the path to advanced echocardiography in the intensive care unit]. / Das EDEC-Curriculum ­ der Weg zur fortgeschrittenen Echokardiographie auf der Intensivstation.

Good knowledge of echocardiography is essential for modern intensive care medicine. A standardized curriculum for acquiring the expertise to perform TTE and TEE is a good way to strengthen one's own diagnostic skills.The EDEC curriculum from ESICM, which has been established for years, offers a good opportunity for structural further training at the advanced level in combination with gaining a high level of professional competence.

[Positive effects of training curriculum using the example of the interdisciplinary intensive care unit]. / Positive Effekte eines Einarbeitungs- und Weiterbildungscurriculums am Beispiel der interdisziplinären Intensivstation.

The training of new medical colleagues in hospitals is due to various aspects such as for instance staff shortages and time pressure often very challenging for everyone involved. Using the example of the interdisciplinary intensive care unit, the positive effects of a structured training curriculum have been scientifically proven in regular employee surveys. There was a statistically significant increase in satisfaction in the quality of induction (p < 0.0001), quality of training (p < 0.0001),  preparation for night shifts in the intensive care unit (p < 0.0001) and an improvement in general satisfaction in the clinic (p < 0.003) can also be shown. For these reasons, such curriculum contributes to increasing the quality of care and patient safety as well as the safety of medical staff in the medical work.

Partial resection of native and prosthetic arteriovenous fistula as a treatment of late infections complications - series of case reports.

INTRODUCTION: Infection of arteriovenous fistula (AV) used for hemodialysis (HD) is associated with massive bleeding, sepsis development, formation of metastatic infectious foci, and a high risk of AV loss. Urgent management of an infected AV is crucial for successful treatment and AV salvage. CASE REPORTS: We present the use of partial resection as a successful method of dealing with late AV infection in two cases. In case 1, the resection was performed due to an infection of the native arteriovenous fistula (AVF) with two defects above the drainage vein aneurysms. In case 2, partial resection and replacement of the prosthetic arteriovenous fistula (AVG) were done due to an infection of HD puncture site. The AVs remained patent in both cases, with no further signs of infection postoperatively and, most importantly, without the need to use a temporary HD catheter. CONCLUSION: The establishment of a new AV is limited by the quality of the venous and arterial systems. All surgical, interventional and non-surgical means should be used to safely maintain the created AV patent. Provided that the requirements of an early indication are met, partial resection of the AV is the method of choice for AV infections and allows us to avoid using a permanent dialysis catheter in our patients.

[Neoadjuvant Use of Isolated Limb Perfusion in Large Myxoid Liposarcoma of the Thigh: a Case Report]. / Neoadjuvantní uzití izolované perfuze koncetiny u objemného myxoidního liposarkomu stehna: kazuistika.

Large unresectable STS presents a therapeutic challenge. Several options are being explored to avoid amputation without compromising the oncological outcome. Neoadjuvant chemotherapy delivers inconsistent and rather unsatisfactory results, preoperative radiotherapy compromises healing, hence it can impede adjuvant systemic treatment. We present a case report of neoadjuvant use of isolated limb perfusion with TNF-alfa and Alkeran (Melphalan) in a patient with initially unresectable large myxoid liposarcoma of the thigh. We achieved 55% reduction in size of the tumor that allowed for wide resection with a safe margin. Pathology confirmed 99% tumor necrosis. The patient has a full function of his extremity and is disease-free at one year follow-up. ILP should be considered as a treatment option which, in selected cases, can contribute to limb sparing surgery. Key words: sarcoma, soft tissue, regional perfusion, chemotherapy, surgery, orthopedic, limb salvage.

PD-1, PD-L1 and PD-L2 Expression in Mantle Cell Lymphoma and Healthy Population.

Cell surface expression of PD-1, PD-L1 and PD-L2 immune checkpoints on B and T cells obtained from patients with mantle cell lymphoma shows ambiguous results across many studies and creates obstacles for the implementation of immune checkpoint inhibitors into the therapy of mantle cell lymphoma. Using multiparameter flow cytometry we analysed surface expression of PD-1, PD-L1 and PD-L2 molecules on B and T cells of 31 newly diagnosed mantle cell lymphomas and compared it with the results of 26 newly diagnosed chronic lymphocytic leukaemias and 20 healthy volunteers. To gain insight into the age-dependent changes of surface expression of these immune checkpoints, flow cytometric subanalysis of 30 healthy volunteers of 25-93 years of age was conducted. Overall, we demonstrated weak surface expression of PD-1, PD-L1 and PD-L2 on B and T cells of mantle cell lymphoma patients (< 10 % when compared to healthy individuals). A significant age-dependent increase in the expression of PD-1 and its ligand PD-L2 was observed in healthy volunteers. Our results suggest that neither PD-1 nor its ligands represent relevant druggable targets for the therapy of mantle cell lymphoma. The observed age-dependent changes in healthy population could impact efficiency of immune checkpoint inhibitors and could be at least partly connected with increased incidence of cancer with age.

Development of vascular substitutes for low-flow peripheral bypass grafting - a review.

The development of a low-flow vascular prosthesis is a very topical issue. The authors present a pathway for the development of a prosthesis with optimal properties based on the idea of mimicking the characteristics of a biological model (saphenous vein graft) and programming these properties in the model of the prosthetic substitute. The vascular prosthesis presented consists of three layers - a non-absorbable scaffold representing vascular “media”, and two absorbable collagen layers - pseudointima and pseudoadventitia. The basic methods of physical testing are presented - the single axis stretch test and inflation-extension test, as well as other procedures that affect the final properties. These include collagen curing, antithrombotic treatment of the inner layer and the use of sterilization methods. The designed new graft was successfully implanted in an ovine model.

The influence of purification of carp collagen used in a novel composite graft with sandwich construction of the wall on its biological properties and graft patency rates.

We compared graft outcome between two types of a novel composite three-layer carp-collagen-coated vascular graft in low-flow conditions in a sheep model. Collagen in group A underwent more cycles of purification than in group B in order to increase the ratio between collagen and residual fat. The grafts were implanted end-to-side in both carotid arteries in sheep (14 grafts in 7 sheep in group A, 18 grafts in 9 sheep in group B) and artificially stenosed on the right side. The flow in the grafts in group A decreased from 297±118 ml/min to 158±159 ml/min (p=0.041) after placement of the artificial stenosis in group A, and from 330±164ml/min to 97±29 ml/min (p=0.0052) in group B (p=0.27 between the groups). From the five surviving animals in group A, both grafts occluded in one animal 3 and 14 days after implantation. In group B, from the six surviving animals, only one graft on the left side remained patent (p=0.0017). Histology showed degradation of the intimal layer in the center with endothelization from the periphery in group A and formation of thick fibrous intimal layer in group B. We conclude that the ratio between collagen and lipid content in the novel three-layer graft plays a critical role in its patency and structural changes in vivo.

Neoadjuvant Hypertermic Isolated Limb Perfusion in Treatment of Undifferentiated Spindle Cell Sarcoma of Lower Limb with Achieved Complete Pathologic Response.

BACKGROUND: Hyperthermic isolated limb perfusion uses therapeutic effect of hyperthermia in the bounded compartment of the limb together with increased concentration of chemotherapy effect than what would be achieved in systemic application. Gold standard was melphalan (Alkeran) in combination with tasonermin (Beromun, tumor necrosis factor alpha). The efficacy of this combination has been demonstrated in limb soft tissue sarcomas and in patients with limb isolated bulky disease of malignant melanoma. CASE: We describe a case of a 65-year-old female patient with undifferentiated spindle-cell sarcoma treated by a multidisciplinary team at the 2nd Surgical Clinic of Cardiovascular Surgery and Clinic of Oncology General University Hospital in Prague and at the Department of Orthopaedics Na Bulovce Hospital with the aim of preserving the limb despite the advanced disease. The patient underwent hyperthermic isolated limb perfusion with tasonermin and melphalan with partial response on magnetic resonance imaging. Subsequent wide resection was done with complete pathological remission according to histological examination maintaining a fully functional limb. The patient is followed without signs of recurrence. CONCLUSION: Hyperthermic isolated limb perfusion with tasonermin and melphalan is an important part of a multimodal approach in the treatment of extremity sarcomas with a high percentage of responses that increase the percentage of limbs retaining resections. Multidisciplinary team should consider this option in patients with localized limb sarcomas and should be performed in specialized centers with experience in this procedure. This work was supported by project Progres Q28-LF1. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.

Visual cortex neurons phase-lock selectively to subsets of LFP oscillations.

It is generally thought that apart from receptive field differences, such as preferred orientation and spatial frequency selectivity, primary visual cortex neurons are functionally similar to each other. However, the genetic diversity of cortical neurons plus the existence of inputs additional to those required to explain known receptive field properties might suggest otherwise. Here we report the existence of desynchronized states in anesthetized cat area 17 lasting up to 45 min, characterized by variable narrow-band local field potential (LFP) oscillations in the range 2-100 Hz and the absence of a synchronized 1/f frequency spectrum. During these periods, spontaneously active neurons phase-locked to variable subsets of LFP oscillations. Individual neurons often ignored frequencies that others phase-locked to. We suggest that these desynchronized periods may correspond to REM sleep-like episodes occurring under anesthesia. Frequency-selective codes may be used for signaling during these periods. Hence frequency-selective combination and frequency-labeled pathways may represent a previously unsuspected dimension of cortical organization. NEW & NOTEWORTHY We investigated spontaneous neuronal firing during periods of desynchronized local field potential (LFP) activity, resembling REM sleep, in anesthetized cats. During these periods, neurons synchronized their spikes to specific phases of multiple LFP frequency components, with some neurons ignoring frequencies that others were synchronized to. Some neurons fired at phase alignments of frequency pairs, thereby acting as phase coincidence detectors. These results suggest that internal brain signaling may use frequency combination codes to generate temporally structured spike trains.

[Weaning from dialysis after acute kidney injury in chronically critically ill]. / Renale Rekompensation nach akuter Nierenschädigung bei chronisch kritisch kranken Patienten.

This study describes the course of renal recovery after dialysis in a specific population of chronically critically ill patients with a history of prolonged and complicated treatment in an intensive care unit. This study shows that, in a specialized center, patients can be successfully weaned from dialysis even months after acute kidney injury (AKI). Of the patients who could be recompensated (33%), approximately 20% achieved renal recovery more than 3 months after the start of dialysis. The duration of renal recovery after AKI did not differ between those patients with pre-existing chronic kidney disease (CKD) and those without. The reason for dialysis treatment such as sepsis, surgery, resuscitation, as well as the risk factors (e. g., diabetes mellitus, arterial hypertension, arteriosclerosis) did not reveal a difference in weaning in a hazard analysis. As a potential risk factor, only age significantly influenced weaning from dialysis in the multivariate hazard model.

Concomitant embolism in the subclavian and pulmonary artery.

INTRODUCTION: Pulmonary embolism is a life-threatening condition that causes obstruction of the pulmonary arteries by an embolus, most often originating from the venous system of the lower limbs or pelvic veins. Depending on the extent of the embolism, an acute right-sided heart failure may result, with subsequent death. Paradoxical embolism is a condition in which a venous thrombus is embolized into the systemic circulation arteries by a right-to-left heart shunt. This condition most commonly occurs in the interatrial septum. The aim of treatment for pulmonary embolism is to remove the obstruction of the lung. Depending on the extent of the embolism and the patients hemodynamic status, thrombolytic or surgical treatment can be chosen. Thrombolytic therapy has become the basis of treatment for all types of acute pulmonary embolism. Currently, open surgery is indicated in hemodynamically unstable patients with massive pulmonary embolism, and increasingly frequently in submassive embolism where thrombolytic therapy is not effective or contraindicated. CASE REPORT: The case report describes a less common condition of pulmonary and concurrently systemic embolization in the subclavian artery. Thrombolytic therapy was contraindicated in this case due to the high risk of secondary embolism to the brain. Surgical embolectomy was performed from the pulmonary arteries simultaneously with direct embolectomy from the subclavian and brachiocephalic artery. The surgical management of acute pulmonary embolism is a suitable method of treatment for both massive pulmonary embolism where the procedure is performed as a life-saving indication, and submassive embolism where less invasive treatment would not be effective. CONCLUSION: Direct embolectomy of subclavian and brachiocephalic artery is associated with minimal risk of iatrogenic embolism into the arteries supplying the brain compared to indirect embolectomy performed from the cubital or brachial artery. Key words: embolism paradoxical embolization - embolectomy foramen ovale stroke.

[Isolated Perfusion of the Upper Extremity with TNF-α - Double Venous Cannulation]. / Izolovaná perfuze horní koncetiny s TNF-α - dvojitá kanylace zilního systému.

BACKGROUND: The authors present a technical variation of the standard cannulation for cardiopulmonary bypass perfusion during hyperthermic isolated limb perfusion (ILP) procedures in selected patients with unresectable soft tissue sarcoma or malignant melanoma. PATIENTS: Of 55 ILP procedures performed at our institution since the procedure was established in 2009, nine were performed at the upper extremity. Standard single venous cannulation was used in five cases, and extended, double venous cannulation in the last four. The standard technique for brachial vein cannulation in a small compartment of the upper extremity entails a problematic and longer perfusion of the upper extremity. This is due to the lower flow rate in the venous system and relatively large surface area with respect to weight. We present a simple technique based on a "Y" cannulation of the venous system via the deep brachial vein and superficial venous system via the basilic vein, delivering a 20% increase in flow rate in the extracorporeal circulation. Faster heating of the upper extremity and a stable thermal environment throughout upper-extremity ILP are essential for successful treatment. CONCLUSION: Extended technique of venous cannulation for extracorporeal circulation setting, due to their advantages, became standard in the upper limb ILP procedure at our institution.Key words: isolated limb perfusion - malignant melanoma - soft tissue sarcoma - upper limb - extracorporeal circulation The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 8. 1. 2017Accepted: 15. 1. 2017.

[Hyperthermic Isolated Limb Perfusion Combined with Tasonermin - a Perfusion Leakage Monitoring Technique]. / Hypertermická izolovaná perfuze koncetin v kombinaci s tasonerminem - technika monitorování úniku perfuzátu.

BACKGROUND: Hyperthermic isolated limb perfusion is used to treat irresectable extremity malignancies. It is based on the following principle - the perfusion of the extremity is isolated from systemic circulation and connected to an extra-corporal circuit via which a very high concentration of a chemotherapeutic agent is administered into the blood compartment of the extremity. In some cases, treatment efficiency can be improved using tasonermin (a TNF-α agent). By itself, tasonermin can cause severe health complications in patients if leakage into systemic circulation results in a level that exceeds the maximally tolerated dose. Therefore, it is important to monitor for leakage during the whole operation. METHOD: Leakage monitoring was performed by a nuclear medicine method based on the measurement of activity of a gamma-emitting radiotracer detected by a scintillation probe located over the heart. An amount of radiotracer that resulted in a basal level of measured signal was first administered into the systemic circuit followed by the administration of a second, one order of magnitude higher amount of radiotracer into the perfusion circuit. Leakage, when it occurred, increased the count rate detected over the heart, and the mathematical relation between leakage level and count rate increase was derived. RESULTS: In our department, the method was tested and optimized during isolated limb perfusion without using a TNF-α agent. Then, accreditation for the use of TNF-α was granted. Since then, the method has been used to monitor leakage in all cases of isolated limb perfusion with TNF-α. All isolated limb perfusion operations with TNF-α passed without complications. The radiation burden was almost negligible for both the patient and medical staff. CONCLUSION: The method described in this report represents a reliable method for perfusion leakage monitoring when using TNF-α in our department.Key words: perfusion - isolated limb - TNF-α - leakage - monitoring - nuclear medicine - radiopharmaceuticalsThe authors declare they have no potential confl icts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 16. 6. 2016Accepted: 21. 6. 2016.

The ratio of diameters between the target artery and the bypass modifies hemodynamic parameters related to intimal hyperplasia in the distal end-to-side anastomosis.

Hemodynamics in the distal end-to-side anastomosis is related to early development of intimal hyperplasia and bypass failure. In this study we investigated the effect of diameter ratios between the target artery and the bypass at three different angles of the connection. The pulsatile flow field was visualized using particle image velocimetry in transparent models with three different angles of the connection (25°, 45°, 60°) and the diameter ratio between the bypass and the target artery was 4.6 mm : 6 mm, 6 mm : 6 mm, and 7.5 mm : 6 mm. Six parameters including location and oscillation of the stagnation point, local energy dissipation, wall shear stress (WSS), oscillatory shear index, spatial and temporal gradient of WSS and their distribution in the target artery were calculated from the flow field. In the wider bypass, the stagnation point oscillated in a greater range and was located more proximal to the anastomosis. Energy dissipation was minimal in a wider bypass with a more acute angle. The maximum WSS values were tree times greater in a narrow bypass and concentrated in a smaller circular region at the floor of the anastomosis. The oscillatory shear index increased with wider bypass and more acute angle. The maximum of spatial gradient of WSS concentrated around the floor and toe of the anastomosis and decreased with more acute angle and wider bypass, the temporal gradient of WSS was stretched more towards the side wall. Greater bypass to target vessel ratio and more acute anastomosis angle promote hemodynamics known to reduce formation of intimal hyperplasia.

Evaluation of cerebrovascular reserve in patients undergoing carotid artery stenting and its usefulness in predicting significant hemodynamic changes during temporary carotid occlusion.

We investigated the usefulness of cerebrovascular reserve (CVR) testing to predict severe hemodynamic changes during proximally protected carotid artery stenting. Of 90 patients referred, 63 eligible underwent complete evaluation of the extent of carotid artery disease and transcranial Doppler ultrasound (TCD) assessment of CVR by means of a breath-holding test and ophthalmic artery flow pattern evaluation. Periprocedural TCD monitoring of the ipsilateral middle cerebral artery flow was performed in 24 patients undergoing proximally protected procedure (requiring induction of flow arrest within internal carotid artery). Abnormal CVR was significantly less common in patients with unilateral compared to bilateral carotid artery disease (26.3 % vs. 76.9 %, p=0.02), while ophthalmic artery flow reversal was rare in patients with unilateral carotid artery disease (2.5 % vs. 42.9 %, p<0.01). During the induction of carotid flow arrest, the average mean flow velocity drop following external carotid artery occlusion was low (3.5 %, p=0.67) compared to the induction of complete flow arrest (32.8 %, p<0.01). Six patients had a total mean flow velocity drop >50 %, including 2 patients with normal pre-procedural CVR. Our results suggest that TCD evaluation of CVR is not a reliable predictor of hemodynamic changes induced during proximally protected carotid artery stenting in patients with unilateral carotid artery disease.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

[Small Molecules in the Treatment of Chronic Lymphocytic Leukemia in 2015 and in the Near Future]. / Malé molekuly v lécbe chronické lymfocytární leukemie v roce 2015 a v blízké budoucnosti.

Chronic lymphocytic leukemia is one of the most common lymphoid malignancies and is characterized by a highly heterogeneous clinical course. Combined regimens, such as fludarabine, cyclophosphamide, and rituximab have led to improvements in survival in younger patients with chronic lymphocytic leukemia and have become the standard of care in fit patients. However, the majority of chronic lymphocytic leukemia patients are elderly and not all patients are eligible for aggressive chemoimmunotherapy. In addition, patients with poor- risk cytogenetics have inferior responses to standard treatments with often shorter durations of response. Furthermore, the treatment outcomes of refractory disease are dismal. Nevertheless, there has been a dramatic change in therapeutic paradigms in the past year. Several new drugs have been approved for the treatment of chronic lymphocytic leukemia, including ibrutinib and idelalisib. These new molecules are orally active agents and both target the B -cell receptor associated kinases. Ibrutinib is a selective, irreversible inhibitor of Bruton tyrosine kinase, whereas idelalisib selectively targets phosphatidylinositol 3- kinase (PI3K) δ isoform. These agents have demonstrated remarkable activity in patients with relapsed/ refractory chronic lymphocytic leukemia, as well as patients with high-risk deletion of the 17p chromosome and/ or TP53 mutation. This review focuses on some of the novel small molecules that are currently approved or in advanced clinical development.

Transcranial Doppler Ultrasound in the Current Era of Carotid Artery Stenting.

Since its introduction in 1982, transcranial Doppler ultrasound (TCD) has become an important diagnostic and monitoring tool and its usefulness has been well established in many clinical applications. In carotid artery stenting (CAS), TCD has mostly been reserved for the optimization of emboli protection devices. Currently, with increasing use of proximal protection systems resembling surgical clamps, TCD has become invaluable in providing the operator an insight into a patient's cerebral hemodynamic status. Additionally, in selected patients, adverse peri- or post-procedural cerebral outcomes may even be predicted allowing the operator to adjust the therapeutic strategy. This review summarizes the current knowledge regarding the use of TCD in patients undergoing CAS and suggests potential directions of future research.