Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats.

OBJECTIVES AND DESIGN: At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. MATERIAL AND METHODS: Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. RESULTS: Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. CONCLUSION: Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts.

Comparison of Different Thawing Protocols in Human Cryopreserved Venous Grafts.

BACKGROUND: The aim of our study was to assess the impact of different thawing protocols on morphological changes arising in cryopreserved human saphenous vein grafts. METHODS: The study was performed in 12 saphenous vein grafts harvested in brain death donors. Storage in the vapor phase of liquid nitrogen for 3 or 5 years followed. Two thawing protocols were tested: slow thawing in a refrigerator at temperature +4°C for 2 hr and rapid thawing-in a water bath at +37°C. Grafts were processed for scanning electron microscopy. Comparisons of continuous parameters under study between experimental groups were performed using the t-test (age, cold ischemia time, exposure to cryoprotectant, time of storage, total thawing time, mean thawing rate, morphology scoring of thawed HSVG) and the median test (HSVG length). Categorical parameters (sex and blood group) were formally tested using the chi-square test. RESULTS: All samples were evaluated according to morphological changes and scored in terms of morphologically intact endothelium, confluent endothelium with structural inhomogeneity, disruption of the intercellular contacts, separation of the endothelial cells, complete loss of the endothelium, and damage of the subendothelial layers. There is no statistically significant difference between the sample sets at the significance level of 0.05. There was no association with donors' age, sex, and time of storage. CONCLUSIONS: Human cryopreserved saphenous vein grafts in our experimental work showed no difference in terms of structural deterioration of the endothelial surface and basal membrane depending on different thawing protocols used.

Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats.

OBJECTIVES AND DESIGN: The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts. MATERIALS AND METHODS: Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0.2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies. RESULTS: The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20.7±6.7 cells, P<0.001) compared to both immunosuppressed groups (TAC1 5.9±5.5 cells, TAC7 6.1±5.1 cells). Day 30 sera from the allogeneic non-immunosuppressed animals decreased significantly the binding of fluorescence-labelled MHC class I (46.9±19.4%) and class II (65.8±11.9%) antibody to donors spleen cells compared with day 30 sera from both immunosuppressed groups (TAC1, anti-MHC class I 102.4±4.2%, p < 0.001, anti-MHC class II 102.6±6.0%), (TAC7, anti-MHC class I 79.9±3.3%, p < 0.001, anti-MHC class II 80.9±2.7%). CONCLUSION: Both immunosuppressed protocols with tacrolimus (administration from day 1 or from day 7 following transplantation) were able to suppress acute cell- and antibody-mediated rejection of cryopreserved abdominal aortic allografts processed in accordance with our new standardized clinical protocol.

Prognostic significance of macrophage invasion in hilar cholangiocarcinoma.

BACKGROUND: Tumor-associated macrophages (TAMs) promote tumor progression and have an effect on survival in human cancer. However, little is known regarding their influence on tumor progression and prognosis in human hilar cholangiocarcinoma. METHODS: We analyzed surgically resected tumor specimens of hilar cholangiocarcinoma (n = 47) for distribution and localization of TAMs, as defined by expression of CD68. Abundance of TAMs was correlated with clinicopathologic characteristics, tumor recurrence and patients' survival. Statistical analysis was performed using SPSS software. RESULTS: Patients with high density of TAMs in tumor invasive front (TIF) showed significantly higher local and overall tumor recurrence (both ρ < 0.05). Furthermore, high density of TAMs was associated with decreased overall (one-year 83.6% vs. 75.1%; three-year 61.3% vs. 42.4%; both ρ < 0.05) and recurrence-free survival (one-year 93.9% vs. 57.4%; three-year 59.8% vs. 26.2%; both ρ < 0.05). TAMs in TIF and tumor recurrence, were confirmed as the only independent prognostic variables in the multivariate survival analysis (all ρ < 0.05). CONCLUSIONS: Overall survival and recurrence free survival of patients with hilar cholangiocarcinoma significantly improved in patients with low levels of TAMs in the area of TIF, when compared to those with a high density of TAMs. These observations suggest their utilization as valuable prognostic markers in routine histopathologic evaluation, and might indicate future therapeutic approaches by targeting TAMs.

Early prediction of survival after open surgical repair of ruptured abdominal aortic aneurysms.

BACKGROUND: Scoring models are widely established in the intensive care unit (ICU). However, the importance in patients with ruptured abdominal aortic aneurysm (RAAA) remains unclear. Our aim was to analyze scoring systems as predictors of survival in patients undergoing open surgical repair (OSR) for RAAA. METHODS: This is a retrospective study in critically ill patients in a surgical ICU at a university hospital. Sixty-eight patients with RAAA were treated between February 2005 and June 2013. Serial measurements of Sequential Organ Failure Assessment score (SOFA), Simplified Acute Physiology Score II (SAPS II) and Simplified Therapeutic Intervention Scoring System-28 (TISS-28) were evaluated with respect to in-hospital mortality. Eleven patients had to be excluded from this study because 6 underwent endovascular repair and 5 died before they could be admitted to the ICU. RESULTS: All patients underwent OSR. The initial, highest, and mean of SOFA and SAPS II scores correlated significant with in-hospital mortality. In contrast, TISS-28 was inferior and showed a smaller area under the receiver operating curve. The cut-off point for SOFA showed the best performance in terms of sensitivity and specificity. An initial SOFA score below 9 predicted an in-hospital mortality of 16.2% (95% CI, 4.3-28.1) and a score above 9 predicted an in-hospital mortality of 73.7% (95% CI, 53.8-93.5, p < 0.01). Trend analysis showed the largest effect on SAPS II. When the score increased or was unchanged within the first 48 h (score >45), the in-hospital mortality rate was 85.7% (95% CI, 67.4-100, p < 0.01) versus 31.6% (95% CI, 10.7-52.5, p = 0.01) when it decreased. On multiple regression analysis, only the mean of the SOFA score showed a significant predictive capacity with regards to mortality (odds ratio 1.77; 95% CI, 1.19-2.64; p < 0.01). CONCLUSION: SOFA and SAPS II scores were able to predict in-hospital mortality in RAAA within 48 h after OSR. According to cut-off points, an increase or decrease in SOFA and SAPS II scores improved sensitivity and specificity.

Application of gentamicin-collagen sponge shortened wound healing time after minor amputations in diabetic patients - a prospective, randomised trial.

INTRODUCTION: Diabetic foot infections are frequently polymicrobial. The lower tissue concentration of systemically administered antibiotics in diabetic patients was reported. Collatamp(®)EG (Syntacoll GmbH Saal/Donau, Germany) is a bioabsorbable, gentamicin impregnated collagen spongeused for local treatment. The aim of this randomized trial was to assess influence of gentamicin-collagen sponge applied to a wound on surgical outcomes after minor amputations in diabetic patients. MATERIAL AND METHODS: Fifty diabetic patients indicated for minor amputation in 2009 at our surgery department were included in the study. Patients were pre-operatively randomised into two groups. Twenty-five patients in group A were treated with gentamicin impregnated collagen sponge applied into wound peri-operatively while 25 patients in group B had minor amputation without gentamicin sponge. RESULTS: There was no significant difference in the demographic data, procedures performed, diabetes duration and peripheral vascular disease severity between the groups. The median glycosylated haemoglobin was 6.0% (range: 4.6-9.5%) in group A and 6.2% (range: 4.0-8.4%) in control group B (non-significant). Median TcPO2 level was 44 (range: 13-67) in group A and 48 (range: 11-69) in control group B (non-significant). The median of wound healing duration in group A was 3.0 weeks (range: 1.7-17.1 weeks) compared to 4.9 weeks (range: 2.6-20.0 weeks) in control group B. This was with a statistically significant difference (p < 0.05). CONCLUSIONS: Application of gentamicin impregnated collagen sponge shortened wound healing duration after minor amputations in diabetic patients by almost 2 weeks.

Immunosuppressive protocol with delayed use of low-dose tacrolimus after aortic transplantation suppresses donor-specific anti-MHC class I and class II antibody production in rats.

BACKGROUND: Arterial allografts are used as vascular conduits in the treatment of prosthetic graft infection. Immunosuppression decreases their rupture risk rate. However, immunosuppression can be unprofitable in florid infection. Previously, we confirmed inhibition of cell-mediated destruction of rat aortic grafts by delayed use of tacrolimus. In this work, we studied the influence of this protocol on the antibody-mediated rejection. MATERIAL AND METHODS: Flow cytometry was used for the retrospective analysis of day 0, 14, and 30 sera obtained from Lewis rat recipients of isogeneic fresh infrarenal aortic grafts (group A) or Brown-Norway rat aortic grafts (group B,C,D) for the presence of donor-specific anti-MHC class I and II antibodies. Tacrolimus in daily dose of 0.2 mg/kg was administered from day 1 to day 30 (group C) or from day 7 to day 30 (group D). RESULTS: Inhibition of fluorescence-labeled anti-BN MHC class I and MHC class II antibodies binding to BN-splenocytes was observed only by day 14 and day 30 sera of allogeneic non-immunosuppressed Lewis rats (group B). The day 30 sera significantly decreased anti-MHC I (42±3%) and anti-MHC II antibody binding (56±3%) compared to day 0 (76±9%, p=0.005 and 79±5%, p=0.003, respectively). Deposition of immunoglobulins G into the tunica media was observed only in non-immunosuppressed aortic allografts on day 30. CONCLUSIONS: Fresh aortic allografts induce donor-specific anti-MHC class I and anti-MHC class II antibody production. Delayed administration of tacrolimus completely suppressed antibody production and antibody-mediated destruction of aortic allografts.

Antibody-mediated rejection of arterialised venous allografts is inhibited by immunosuppression in rats.

OBJECTIVES AND DESIGN: We determined in a rat model (1) the presence and dynamics of alloantibodies recognizing MHC complexes on quiescent Brown-Norway (BN) splenic cells in the sera of Lewis (LEW) recipients of Brown-Norway iliolumbar vein grafts under tacrolimus immunosuppression; and (2) the presence of immunoglobulins in the wall of acute rejected vein allografts. MATERIALS AND METHODS: Flow cytometry was used for the analysis of day 0, 14 and 30 sera obtained from Lewis recipients of isogeneic iliolumbar vein grafts (group A) or Brown-Norway grafts (group B, C) for the presence of donor specific anti-MHC class I and II antibodies. Tacrolimus 0.2 mg/kg daily was administered from day 1 to day 30 (group C). Histology was performed on day 30. RESULTS: Sera obtained preoperatively and on day 30 were compared in all groups. The statistically significant decrease of anti MHC class I and II antibody binding was observed only in allogenic non-immunosuppressed group B (splenocytes: MHC class I - day 0 (93% ± 7% ) vs day 30 (66% ± 7%), p = 0.02, MHC class II - day 0 (105% ± 3% ) vs day 30 (83% ± 5%), p = 0.003; B-cells: MHC class I - day 0 (83% ± 5%) vs day 30 (55% ± 6%), p = 0.003, MHC class II - day 0 (101% ± 1%) vs day 30 (79% ± 6%), p = 0.006; T-cells: MHC class I - day 0 (71% ± 7%) vs day 30 (49% ± 5%), p = 0.04). No free clusters of immunoglobulin G deposition were detected in any experimental group. CONCLUSION: Arterialized venous allografts induce strong donor-specific anti-MHC class I and anti-MHC class II antibody production with subsequent immune-mediated destruction of these allografts with no evidence of immunoglobulin G deposition. Low-dose tacrolimus suppress the donor-specific antibody production.

Outcome after open surgery repair in endovascular-suitable patients with ruptured abdominal aortic aneurysms.

BACKGROUND: Endovascular aneurysm repair (EVAR) has been suggested in several studies to be superior to open surgery repair (OSR) for the treatment of ruptured abdominal aortic aneurysms (rAAAs), but this finding might be affected by selection bias based on aneurysm morphology and patient characteristics. We tested rAAA anatomy according to EVAR suitability in patients undergoing OSR to assess the impact on mortality. PATIENTS AND METHODS: This retrospective analysis reports on 83 patients with rAAAs treated between November 2002 and July 2013. Pre-operative computed tomography (CT) scans were evaluated based on EVAR suitability and were determined by blinded independent reviewers. CT scans were lacking due to acquisition in an external institution with no availability (n = 9) or solely ultrasound evaluations (n = 8). In addition patient characteristics and outcomes were assessed. RESULTS: All patients who underwent OSR and who had available preoperative CT scans were included in the study (n = 66). In summary, 42 % of the patients (28/66; 95 % confidence interval [CI], 30.5 - 54.4) were considered eligible for EVAR according to pre-operative CT scans and 58 % of the patients (38/66; 95 % CI, 45.6 - 69.5) were categorized as unsuitable for endovascular repair. Patients suitable for EVAR had a significantly lower prevalence of in-hospital deaths (25 % [7/28]; 95 % CI, 9 - 41) in contrast to patients unsuitable for EVAR (53 % [20/38]; 95 % CI, 36.8 - 68.5; p = 0.02). CONCLUSIONS: EVAR-suitable patients had a highly significant mortality reduction undergoing OSR. Thus, the present study proposes that EVAR suitability is a positive predictor for survival after open repair of rAAA.

Polypropylene mesh repair of incisional hernia after kidney transplantation: single-center experience and review of the literature.

BACKGROUND: Incisional hernia after kidney transplantation increases patient morbidity and impacts quality of life. Reports of hernia mesh repair after kidney transplantation are rare; thus, the benefit of mesh hernioplasty in transplanted patients is assumed. However, it is also assumed that transplant patients are susceptible to incisional and mesh infections. MATERIAL/METHODS: Between January 1, 2005 and December 31, 2010, we performed 1067 kidney transplantations. Twenty-eight patients developed incisional hernias (2.6%), and mesh repair was performed in 20 of them (8 women, 12 men; median age 59.5 years, range 43 to 68 years). We retrospectively studied this latter group. We also reviewed the literature regarding the results of this treatment. RESULTS: Postoperative mortality was zero, but postoperative wound bleeding led to surgical revision in 1 patient. Wound infection did not occur. During the follow-up period we observed 4 hernia recurrences (20%). CONCLUSIONS: In conclusion, our retrospective study and review of the literature confirmed the safety and low incidence (1.1% to 3.8%) of mesh hernia repair in chronic immunosuppressed patients after renal transplantation, which has a minimal risk of wound infection and no higher risk of hernia recurrence than in non-transplant patients.

[Eversion carotid endarterectomy: evaluation of results after changing the operation technique]. / Everzní endarterektomie arterie carotis interna: hodnocení výsledku po zmene operacní techniky.

BACKGROUND: Stroke is the third most common cause of mortality, and carotid artery stenosis causes up to 30% of all ischemic strokes. Eversion CEA (ECEA) has been proposed as an acceptable alternative to the standard bifurcation endarterectomy in many patients. This study was designed to analyze the long-term durability of ECEA in symptomatic and asymptomatic patients. Data were retrospectively reviewed to determine the incidence of major adverse cardiovascular events (MACEs) within 30 days of surgery, late survival, and the incidence of restenosis. METHODS AND RESULTS: From January 1999 to June 2010, 344 ECEAs were performed on 324 patients (34% female, 38% symptomatic). The mean follow-up period was 30 months. CONCLUSIONS: MACEs occurred in 28 patients (8.6%). The overall incidence of stroke or death after ECEA was 1.7% and 0.9% at discharge. The overall incidence of stoke or death after CEA was 4.3% and 21% (14 strokes, 69 deaths, 8 of which were stroke-related). The overall occurrence of any restenosis (> 50%) after CEA was 4.3% (14 of 324 procedures).

Successful surgical treatment of multilevel aortic aneurysms combined with renal transplantation.

The concomitant presence of a thoracic aortic aneurysm and an abdominal aortic aneurysm in patients considered for renal transplantation is extremely rare. To our knowledge, this is the first case report of the successful treatment of multilevel aortic aneurysms together with renal transplantation. The treatment modalities in renal transplant patients with concomitant aortic aneurysms are discussed.

Delayed administration of FK 506 is sufficient to suppress acute rejection changes after aortal transplantation in rats.

Arterial allografts are used now-a-days as a modality in the treatment of vascular prosthesis infections. Prolonged administration of immunosuppressive drugs seemed to be essential for long-time patency rates of alloarterial vascular reconstructions. Nevertheless, the use of immunosuppressives if there exist an acute infection is controversial. The experimental work described herein studied effects of a delayed low-dose FK 506 administration on the development of acute rejection changes 30 days after aortal transplantation in rats. The response of the recipient's immune system to aortal wall antigens of the donor in the field of no immunosuppression resulted in an intimal proliferation and its infiltration by immunocompetent cells of the recipient, necrosis of medial smooth muscle cells, including deposition of immunoglobulins, and a massive adventitial infiltration of CD4 and CD8 positive cells. On the other hand, all the principal histological signs of rejection listed above were suppressed by FK 506 administration, no matter whether the immunosuppressive was administered on day 0 or day 7 after the transplantation.

Renal transplantation in patients with abdominal aortic aneurysm--a new surgical approach.

An increasing number of abdominal aortic aneurysms occurs in renal failure patients because of an accelerated atherosclerosis process associated with uraemia. When technically feasible, endovascular repair of an abdominal aortic lesion should be considered as the treatment of choice. If a surgical repair is suggested, there are several options to select from. Since November 1999, we performed simultaneous aortic reconstruction using fresh arterial allograft and kidney transplantation in five uraemic patients with asymptomatic abdominal aortic aneurysm. The operative and postoperative course of four patients passed without major complications. One patient had ischaemic colitis early after the operation, which required a partial resection of the colon. One patient died 6 weeks after the operation due to non-vascular causes. In conclusion, the advantage of our single-phase procedure is that both diseases are treated simultaneously during a single hospital stay. Moreover, with our procedure, the risk of vascular graft infection in patients with chronic immunosuppression is low.

Serotonin levels in the small bowel mucosa as a marker of ischemic injury during small bowel preservation.

Of the serotonin occurring in the small bowel mucosa, 95% is present in enterochromaffine cells. The cold ischemia during small bowel transplantation results in mucosal injury and releasing of serotonin into the lumen. Because of it, the mucosal concentration of serotonin is decreasing. The aim of our study was to establish the correlation between changes in serotonin levels in small bowel mucosa during grafts preservation and cold ischemic time. Wistar rats (n= 35) weighing 322+/-18g, divided into five main groups (n= 7/group) according to the time of small bowel grafts preservation (0, 1, 6, 9, and 12 hours), were used as experimental animals. The grafts were preserved in 4 degrees C histidine-tryptophane-ketoglutarate (HTK) solution. Tissue samples for mucosal serotonin concentration measurement and for light microscopic evaluation were taken after predefined cold ischemic times. Quantitative histological assessment was made using the Park's small bowel wall injury grading scheme. The t-test for dependent samples was used for statistical analysis. The mean serotonin mucosal concentrations after 0, 1, 6, 9, and 12 hours of cold ischemic injury were 433.09+/-160.33, 402.6+/-120.53, 412.5+/-47.57 ng/mL, 190.8+/-45.88 and 145.2+/-16.78 ng/mL Statistically significant differences (p<0.05) were between 6, 9, and 12 hours of cold ischemia. Morphological changes of small bowel mucosa graded by Park's scheme after the same ischemic intervals were 0, 0.5+/-0.47, 0.97+/-0.41, 1.74+/-0.69, and 1.84+/-0.64. Statistically significant differences (p<0.05) were demonstrated between all preservation times except between 9 and 12 hours of cold ischemia. Morphological changes in small bowel mucosa correlated with cold ischemic time, as well as with serotonin mucosal concentration. These data indicate the possibility of use a serotonin concentration in small bowel mucosa as a parameter of small bowel grafts ischemic injury.

Model of small bowel transplantation with systemic venous drainage in rats.

Authors in details describe microsurgical technique of heterotopic small bowel transplantation (SBT) with systemic venous drainage in rats. Surgical technique was practiced on laboratory Wistar rat (n = 40), male with weight 240-300 g. This paper in details analyzes the surgical procedure and includes methods of anesthesia, organ harvest, methods of conservation, transplantation and their complications. The aim of authors is to describe a reproducible model of small bowel transplantation in rat and to get a basis for next experimental studies, which will investigate others aspects of SBT like preservation-reperfusion injury and immunological aspect as rejection. Five days of surviving of rats was accepted as successful.