Organization model for allotransplantations of cryopreserved vascular grafts in Czech Republic.

The transplantation of fresh or cryopreserved vascular allografts in patients with a prosthetic graft infection or critical limb ischemia is necessary for their limb salvage and, in many cases, represents a lifesaving procedure. While transplantation of fresh allografts has a long history in the Czech Republic, the standard use of cryopreserved vascular allografts was introduced into the clinical practice in 2011 as a result of the implementation of EU Directive 2004/23/EC into national legislation (Human Cell and Tissue Act No. 296/2008 Coll.). The authors present an organizational model based on cooperation between the majority of Czech Transplant Centers with a tissue establishment licensed by the national competent authority. In various points, we are addressing individual aspects of experimental and clinical studies which affect clinical practice. Based on experimental and clinical work, the first validation of cryopreserved arterial and venous grafts for clinical use was performed between 2011 and 2013. The growing number of centers participating in this programme led to a growing number of patients who underwent transplantation of vascular allografts. In 2015 the numbers of transplanted fresh versus cryopreserved allografts in the Czech Republic were almost equal. Cooperation of the participating centers in the Czech Republic with the licensed Tissue Establishment made it possible to achieve a full compliance with the European Union Directives, and harmonized national legal norms and assured a high quality of cryopreserved vascular allografts.

Characterisation and comparison of adipose tissue macrophages from human subcutaneous, visceral and perivascular adipose tissue.

BACKGROUND AND AIMS: Macrophages play important roles in adipose tissue inflammation and its consequences. Unfortunately, a detailed description of the macrophage phenotypes in different human adipose tissues is not available. SUBJECTS AND METHODS: Subcutaneous, visceral and perivascular adipose tissues were obtained from 52 living kidney donors during live donor nephrectomy. Stromal vascular fractions were isolated, and the macrophage phenotypes were analyzed by flow cytometry using surface markers (CD14, CD16, CD36, and CD163). RESULTS: In addition to CD16 positivity, pro-inflammatory macrophages also display high scavenger receptor CD36 expression. The great majority of CD16 negative macrophages express the anti-inflammatory CD163 marker. The presence of pro-inflammatory macrophages was almost twice as high in visceral (p < 0.0001) and perivascular (p < 0.0001) adipose tissues than in subcutaneous tissue. This difference was substantially more pronounced in the postmenopausal women subgroup, consequentlly, the total difference was driven by this subgroup. CONCLUSION: We obtained detailed information about M1 and M2 macrophage phenotypes in human adipose tissue. The visceral and perivascular adipose tissues had substantially higher pro-inflammatory characteristics than the subcutaneous tissue. The higher proportion of pro-inflammatory macrophages in the visceral adipose tissue of postmenopausal women might be related to an increased cardiovascular risk.

The effect of cytokines produced by human adipose tissue on monocyte adhesion to the endothelium.

Visceral adipose tissue (VAT) may play a critical role in atherosclerotic cardiovascular disease. The goal of this study was to determine the effect of human VAT-released pro­inflammatory cytokines on monocyte adhesion to the endothelium. The cytokine effects on monocyte adhesion to the endothelial cells (ECs) were tested using adipose tissue-conditioned media (ATCM) prepared by culturing human VAT. The cytokines concentrations in ATCM, the cytokines expression and adhesion molecules in stimulated ECs were measured. The concentrations of IL-1ß,TNF-α,MCP-1,IL-10,and RANTES measured in ATCM correlated positively with monocyte adhesiveness to ECs. Additionally, ATCM increased the adhesion molecules (ICAM-1, VCAM-1) gene expression. Selective inhibitors highlighted the importance of IL-1ß and TNF-α in the process by a significant decrease in monocyte adhesion compared to ATCM preconditioning without inhibitors. Human VAT significantly increased monocyte adhesion to ECs. It was significantly influenced by IL-1ß, TNF-α, MCP-1, IL-10, and RANTES, with IL-1ß and TNF­α having the strongest impact.

Polarization of Macrophages in Human Adipose Tissue is Related to the Fatty Acid Spectrum in Membrane Phospholipids.

Residential macrophages in adipose tissue play a pivotal role in the development of inflammation not only within this tissue, but also affect the proinflammatory status of the whole body. Data on human adipose tissue inflammation and the role of macrophages are rather scarce. We previously documented that the proportion of proinflammatory macrophages in human adipose tissue correlates closely with non-HDL cholesterol concentrations. We hypothesized that this is due to the identical influence of diet on both parameters and decided to analyze the fatty acid spectrum in cell membrane phospholipids of the same individuals as a parameter of the diet consumed. Proinflammatory and anti-inflammatory macrophages were isolated from human adipose tissue (n = 43) and determined by flow cytometry as CD14+CD16+CD36high and CD14+CD16-CD163+, respectively. The spectrum of fatty acids in phospholipids in the cell membranes of specimens of the same adipose tissue was analyzed, and the proportion of proinflammatory macrophage increased with the proportions of palmitic and palmitoleic acids. Contrariwise, these macrophages decreased with increasing alpha-linolenic acid, total n-3 fatty acids, n-3/n-6 ratio, and eicosatetraenoic acid. A mirror picture was documented for the proportion of anti-inflammatory macrophages. The dietary score, obtained using a food frequency questionnaire, documented a positive relation to proinflammatory macrophages in individuals who consumed predominantly vegetable fat and fish, and individuals who consumed diets based on animal fat without fish and nut consumption. he present data support our hypothesis that macrophage polarization in human visceral adipose tissue is related to fatty acid metabolism, cell membrane composition, and diet consumed. It is suggested that fatty acid metabolism might participate also in inflammation and the risk of developing cardiovascular disease.

The Superiority of Removable Contact Splints in the Healing of Diabetic Foot during Postoperative Care.

OBJECTIVE: Off-loading is one of the crucial components of diabetic foot (DF) therapy. However, there remains a paucity of studies on the most suitable off-loading for DF patients under postoperative care. The aim of our study was to evaluate the effect of different protective off-loading devices on healing and postoperative complications in DF patients following limb preservation surgery. METHODS: This observational study comprised 127 DF patients. All enrolled patients had undergone foot surgery and were off-loaded empirically as follows: wheelchair+removable contact splint (RCS) (group R: 29.2%), wheelchair only (group W: 48%), and wheelchair+removable prefabricated device (group WP: 22.8%). We compared the primary (e.g., the number of healed patients, healing time, and duration of antibiotic (ATB) therapy) and secondary outcomes (e.g., number of reamputations and number and duration of rehospitalizations) with regard to the operation regions across all study groups. RESULTS: The lowest number of postoperative complications (number of reamputations: p = 0.028; rehospitalizations: p = 0.0085; and major amputations: p = 0.02) was in group R compared to groups W and WP. There was a strong trend toward a higher percentage of healed patients (78.4% vs. 55.7% and 65.5%; p = 0.068) over a shorter duration (13.7 vs. 16.5 and 20.3 weeks; p = 0.055) in the R group, as well. Furthermore, our subanalysis revealed better primary outcomes in patients operated in the midfoot and better secondary outcomes in patients after forefoot surgery-odds ratios favouring the R group included healing at 2.5 (95% CI, 1.04-6.15; p = 0.037), reamputations at 0.32 (95% CI, 0.12-0.84; p = 0.018), and rehospitalizations at 0.22 (95% CI, 0.08-0.58; p = 0.0013). CONCLUSIONS: This observational study suggests that removable contact splint combined with a wheelchair is better than a wheelchair with or without removable off-loading device for accelerating wound healing after surgical procedures; it also minimises overall postoperative complications, reducing the number of reamputations by up to 77% and the number of rehospitalizations by up to 66%.

Current vascular allograft procurement, cryopreservation and transplantation techniques in the Czech Republic.

BACKGROUND: Vascular allotransplantations are performed worldwide in selected patients suffering from vascular prosthesis infection or critical limb ischemia. Either fresh or cryopreserved vascular allograft may be used. OBJECTIVES: In various points, we address several aspects (allograft procurement, cryopreservation and transplantation technique) of the program of vascular allotransplantations in the Czech Republic. MATERIAL AND METHODS: Vascular grafts retrieval has been done within multiorgan harvests using no-touch technique. Very short time of cold ischemia is achieved due to close cooperation with Tissue Establishment where the following processing of cryopreservation is performed. Meeting all necessary quality criteria is a prerequisity for releasing grafts for clinical application. Standardized thawing protocol and surgical handling aims to minimize microfractures before implantation. RESULTS: Based on experimental and clinical work, the first validation of cryopreserved arterial and venous grafts for clinical use was performed between 2011 and 2013 in the Czech Republic. The developement of storage of vascular tissue in banks was stimulated in 2000-2010 by the issue of EU directives and national harmonized norms, aimed at assurance of high quality and safety of cells and tissues used for transplantations in humans. CONCLUSIONS: There are several crucial moments affecting final quality, including graft retrieval within a multiorgan harvest, short ischemic time, cryopreservation and thawing technique used. The recommended surgical handling during implantation may also affect results and graft-related complications.

Sharp liver excision under hepatic vascular exclusion in case of liver transplant for large polycystic disease. Case report of a new surgical technique.

INTRODUCTION: Polycystic liver disease is observed in 75-90% of patients with autosomal dominant polycystic kidney disease (ADPKD). ADPKD has a high prevalence of 1/1000. Hepatomegaly severely reduces quality of life and liver transplantation seems to be method of choice for many patients. Because of the rarity of this disease and the small number of symptomatic patients with massive hepatomegaly indicated for the transplantation, there is no standard approach for explantation of the liver. CASE PRESENTATION: In our case, 57-year-old woman with massive hepatomegaly was treated with simultaneous split liver and kidney transplantation with bilateral nephrectomy. DISCUSSION: For the native liver excision we used unique surgical approach - sharp liver transection under hepatic vascular exclusion. Because we experienced some cases with massive bleeding during the polycystic liver explantation, we decided to change the surgical approach. The technique offers limited blood loss and comfortable operation field exposure. CONCLUSION: The giant polycystic liver could safely be explanted only using sharp transection hepatectomy under hepatic vascular exclusion. There is significant difference between blood loss in patients treated with or without hepatic vascular exclusion.

Cardiovascular disease predictors and adipose tissue macrophage polarization: Is there a link?

Background The risk of cardiovascular disease is closely connected to adipose tissue inflammation. The links between cardiovascular risk predictors and pro and anti-inflammatory macrophages in human adipose tissue were analysed to gain an insight into the pathophysiology of cardiovascular disease. Design Subcutaneous and visceral adipose tissues were obtained from 79 subjects, 52 living kidney donors (during nephrectomy) and 27 patients with peripheral artery disease (during arterial tree reconstruction). Methods Macrophage subsets were isolated from adipose tissues and analysed by flow cytometry using CD14, CD16, CD36 and CD163 monoclonal antibodies. The mutually adjusted differences of phagocytic pro-inflammatory (CD14 + CD16 + CD36high), anti-inflammatory (CD14 + CD16-CD163+) and transitional subsets of macrophages were analysed in relation to cardiovascular predictors (sex, age, body mass index, smoking, hypercholesterolaemia, hypertension and statin treatment). Results Age, male sex and hypercholesterolaemia were closely positively associated with the phagocytic pro-inflammatory macrophage subset in visceral adipose tissues. Interestingly, the proportion of phagocytic pro-inflammatory macrophages was relevantly decreased by statin therapy. A strong positive association of body mass index to the phagocytic pro-inflammatory subset was found in subcutaneous adipose tissues only. A minor transitional subpopulation, CD14 + CD16 + CD36lowCD163+, increased with age in both adipose tissues. This transitional subpopulation was also negatively associated with obesity and hypercholesterolaemia in visceral adipose tissues. Conclusion An effect of cardiovascular risk predictors on adipose tissue macrophage subpopulations was revealed. Interestingly, while age, male sex and hypercholesterolaemia were connected with the pro-inflammatory macrophage subpopulation in visceral adipose tissues, body mass index had a prominent effect in subcutaneous adipose tissues only. A decreasing effect of statins on these pro-inflammatory macrophages was documented.

Macrophage subsets in the adipose tissue could be modified by sex and the reproductive age of women.

The presence of proinflammatory monocytes/macrophages (CD14+CD16+) has been documented in conditions of inflammation, such as atherosclerosis. We analysed the proportion of proinflammatory monocytes/macrophages in perirenal and perivascular fat in healthy living kidney donors with regard to sex and age reflecting reproductive status in women; therefore, women were further divided to younger and older group (younger and older than 51 years) reflecting potential age of menopause. Monocyte/macrophages were identified as CD14+ mononuclear cells and divided into subpopulations based on the co-expression of CD16. We found no differences in the monocyte/macrophage content between men (n = 15) and women (n = 28). Conversely, we observed a higher proportion of double positive CD14+CD16+ monocytes/macrophages in older women (n = 14) compared to younger women (n = 14). In addition, a strong correlation was found between the monocyte/macrophage content in fat and age only in older women. Therefore, proinflammatory monocytes/macrophages (CD14+CD16+) should be evaluated according to the sex and age.