Loss of Endothelial Glycocalyx During Normothermic Machine Perfusion of Porcine Kidneys Irrespective of Pressure and Hematocrit.

Normothermic machine perfusion (NMP) is a promising modality for marginal donor kidneys. However, little is known about the effects of NMP on causing endothelial glycocalyx (eGC) injury. This study aims to evaluate the effects of NMP on eGC injury in marginal donor kidneys and whether this is affected by perfusion pressures and hematocrits. Methods: Porcine slaughterhouse kidneys (n = 6/group) underwent 35 min of warm ischemia. Thereafter, the kidneys were preserved with oxygenated hypothermic machine perfusion for 3 h. Subsequently, 4 h of NMP was applied using pressure-controlled perfusion with an autologous blood-based solution containing either 12%, 24%, or 36% hematocrit. Pressures of 55, 75, and 95 mm Hg were applied in the 24% group. Perfusate, urine, and biopsy samples were collected to determine both injury and functional parameters. Results: During NMP, hyaluronan levels in the perfusate increased significantly (P < 0.0001). In addition, the positivity of glyco-stained glycocalyx decreased significantly over time, both in the glomeruli (P = 0.024) and peritubular capillaries (P = 0.003). The number of endothelial cells did not change during NMP (P = 0.157), whereas glomerular endothelial expression of vascular endothelial growth factor receptor-2 decreased significantly (P < 0.001). Microthrombi formation was significantly increased after NMP. The use of different pressures and hematocrits did not affect functional parameters during perfusion. Conclusions: NMP is accompanied with eGC and vascular endothelial growth factor receptor-2 loss, without significant loss of endothelial cells. eGC loss was not affected by the different pressures and hematocrits used. It remains unclear whether endothelial injury during NMP has harmful consequences for the transplanted kidney.

Renal Endothelial Cytotoxicity Assay to Diagnose and Monitor Renal Transplant Recipients for Anti-Endothelial Antibodies.

Tissue-specific nonhuman leukocyte antigen (HLA) antigens can play crucial roles in allograft immunity and have been shown to trigger humoral responses leading to rejection of HLA-matched kidney allografts. Interest in the role of endothelial-specific antigens has grown over the past years, and several case reports have been described in which antibodies reacting with endothelial cells (ECs) are associated with rejection. Such antibodies escape the detection in conventional crossmatch tests as they do not react with lymphocytes. However, due to the heterogeneity of endothelial cells from different vascular beds, it remains difficult to draw organ-specific conclusions from studies describing endothelial crossmatch assays. We present a case of a 69-year-old male patient whose kidney allograft was rejected as hyperacute, despite the absence of pretransplant HLA-specific antibodies. To place findings from previous studies in a kidney-related context, we performed crossmatch assays with primary renal endothelial cells. The patient's serum was reactive with primary renal ECs, demonstrated by antibody binding and complement-dependent cytotoxicity. Antibodies from this patient did not react with lymphocytes nor were HLA donor-specific antibodies (DSAs) found. Two years later, the patient successfully received a second kidney transplant after treatment with rituximab and plasmapheresis before and after transplantation. We demonstrated that the removal of antibodies against non-HLA EC-specific molecules can be monitored using a primary renal EC crossmatch test, possibly contributing to a successful transplantation outcome.

Antigen and Cell-Based Assays for the Detection of Non-HLA Antibodies.

To date, human leukocyte antigens (HLA) have been the major focus in the approach to acute and chronic antibody-mediated rejection (AMBR) in solid-organ transplantation. However, evidence from the clinic and published studies has shown that non-HLA antibodies, particularly anti-endothelial cell antibodies (AECAs), are found either in the context of AMBR or synergistically in the presence of donor-specific anti-HLA antibodies (DSA). Numerous studies have explored the influence of AECAs on clinical outcomes, yet the determination of the exact clinical relevance of non-HLA antibodies in organ transplantation is not fully established. This is due to highly heterogeneous study designs including differences in testing methods and outcome measures. Efforts to develop reliable and sensitive diagnostic non-HLA antibody tests are continuously made. This is essential considering the technical difficulties of non-HLA antibody assays and the large variation in reported incidences of antibodies. In addition, it is important to take donor specificity into account in order to draw clinically relevant conclusions from non-HLA antibody assays. Here, we provide an overview of non-HLA solid-phase and cell-based crossmatch assays for use in solid-organ transplantation that are currently available, either in a research setting or commercially.

Weak Expression of Terminal Complement in Active Antibody-Mediated Rejection of the Kidney.

Background: The role of the complement system in antibody-mediated rejection (ABMR) is insufficiently understood. We aimed to investigate the role of local and systemic complement activation in active (aABMR). We quantified complement activation markers, C3, C3d, and C5b-9 in plasma of aABMR, and acute T-cell mediated rejection (aTCMR), and non-rejection kidney transplant recipients. Intra-renal complement markers were analyzed as C4d, C3d, C5b-9, and CD59 deposition. We examined in vitro complement activation and CD59 expression on renal endothelial cells upon incubation with human leukocyte antigen antibodies. Methods: We included 50 kidney transplant recipients, who we histopathologically classified as aABMR (n=17), aTCMR (n=18), and non-rejection patients (n=15). Results: Complement activation in plasma did not differ across groups. C3d and C4d deposition were discriminative for aABMR diagnosis. Particularly, C3d deposition was stronger in glomerular (P<0,01), and peritubular capillaries (P<0,05) comparing aABMR to aTCMR rejection and non-rejection biopsies. In contrast to C3d, C5b-9 was only mildly expressed across all groups. For C5b-9, no significant difference between aABMR and non-rejection biopsies regarding peritubular and glomerular C5b-9 deposition was evident. We replicated these findings in vitro using renal endothelial cells and found complement pathway activation with C4d and C3d, but without terminal C5b-9 deposition. Complement regulator CD59 was variably present in biopsies and constitutively expressed on renal endothelial cells in vitro. Conclusion: Our results indicate that terminal complement might only play a minor role in late aABMR, possibly indicating the need to re-evaluate the applicability of terminal complement inhibitors as treatment for aABMR.

Machine-perfused donor kidneys as a source of human renal endothelial cells.

Renal endothelial cells (ECs) play crucial roles in vasorelaxation, ultrafiltration, and selective transport of electrolytes and water, but also in leakage of the glomerular filtration barrier and inflammatory processes like complement activation and leukocyte recruitment. In addition, they are target cells for both cellular and antibody-mediated rejection in the transplanted kidney. To study the molecular and cellular processes underlying EC behavior in renal disease, well-characterized primary renal ECs are indispensible. In this report, we describe a straightforward procedure to isolate ECs from the perfusion fluid of human donor kidneys by a combination of negative selection of monocytes/macrophages, positive selection by CD31 Dynabeads, and propagation in endothelium-specific culture medium. Thus, we isolated and propagated renal ECs from 102 donor kidneys, representative of all blood groups and major human leukocyte antigen (HLA) class I and II antigens. The obtained ECs were positive for CD31 and von Willebrand factor, expressed other endothelial markers such as CD34, VEGF receptor-2, TIE2, and plasmalemmal vesicle associated protein-1 to a variable extent, and were negative for the monocyte marker CD14 and lymphatic endothelial marker podoplanin. HLA class II was either constitutively expressed or could be induced by interferon-γ. Furthermore, as a proof of principle, we showed the diagnostic value of this renal endothelial biobank in renal endothelium-specific cross-matching tests for HLA antibodies.NEW & NOTEWORTHY We describe a new and widely accessible approach to obtain human primary renal endothelial cells in a standardized fashion, by isolating from the perfusate of machine-perfused donor kidneys. Characterization of the cells showed a mixed population originating from different compartments of the kidney. As a proof of principle, we demonstrated a possible diagnostic application in an endothelium-specific cross-match. Next to transplantation, we foresee further applications in the field renal endothelial research.

Plasma cadmium is associated with increased risk of long-term kidney graft failure.

The kidney is one of the most sensitive organs to cadmium-induced toxicity, particularly in conditions of long-term oxidative stress. We hypothesized that, in kidney transplant recipients, nephrotoxic exposure to cadmium represents an overlooked hazard for optimal graft function. To test this, we performed a prospective cohort study and included 672 outpatient kidney transplant recipients with a functioning graft of beyond one year. The median plasma cadmium was 58 ng/L. During a median 4.9 years of follow-up, 78 kidney transplant recipients developed graft failure with a significantly different distribution across tertiles of plasma cadmium (13, 26, and 39 events, respectively). Plasma cadmium was associated with an increased risk of graft failure (hazard ratio 1.96, 95% confidence interval 1.56‒2.47 per log2 ng/L). Similarly, a dose-response relationship was observed over increasing tertiles of plasma cadmium, after adjustments for potential confounders (donor, recipient, transplant and lifestyle characteristics), robust in both competing risk and sensitivity analyses. These findings were also consistent for kidney function decline (graft failure or doubling of serum creatinine). Thus, plasma cadmium is independently associated with an increased risk of long-term kidney graft failure and decline in kidney function. Further studies are needed to investigate whether exposure to cadmium represents an otherwise overlooked modifiable risk factor for adverse long-term graft outcomes in different populations.

Properdin Pattern Recognition on Proximal Tubular Cells Is Heparan Sulfate/Syndecan-1 but Not C3b Dependent and Can Be Blocked by Tick Protein Salp20.

Introduction: Proteinuria contributes to progression of renal damage, partly by complement activation on proximal tubular epithelial cells. By pattern recognition, properdin has shown to bind to heparan sulfate proteoglycans on tubular epithelium and can initiate the alternative complement pathway (AP). Properdin however, also binds to C3b(Bb) and properdin binding to tubular cells might be influenced by the presence of C3b(Bb) on tubular cells and/or by variability in properdin proteins in vitro. In this study we carefully evaluated the specificity of the properdin - heparan sulfate interaction and whether this interaction could be exploited in order to block alternative complement activation. Methods: Binding of various properdin preparations to proximal tubular epithelial cells (PTEC) and subsequent AP activation was determined in the presence or absence of C3 inhibitor Compstatin and properdin inhibitor Salp20. Heparan sulfate proteoglycan dependency of the pattern recognition of properdin was evaluated on PTEC knocked down for syndecan-1 by shRNA technology. Solid phase binding assays were used to evaluate the effectivity of heparin(oids) and recombinant Salp20 to block the pattern recognition of properdin. Results: Binding of serum-derived and recombinant properdin preparations to PTECs could be dose-dependently inhibited (P < 0.01) and competed off (P < 0.01) by recombinant Salp20 (IC50: ~125 ng/ml) but not by Compstatin. Subsequent properdin-mediated AP activation on PTECs could be inhibited by Compstatin (P < 0.01) and blocked by recombinant Salp20 (P < 0.05). Syndecan-1 deficiency in PTECs resulted in a ~75% reduction of properdin binding (P = 0.057). In solid-phase binding assays, properdin binding to C3b could be dose-dependently inhibited by recombinant Salp20> heparin(oid) > C3b. Discussion: In this study we showed that all properdin preparations recognize heparan sulfate/syndecan-1 on PTECs with and without Compstatin C3 blocking conditions. In contrast to Compstatin, recombinant Salp20 prevents heparan sulfate pattern recognition by properdin on PTECs. Both complement inhibitors prevented properdin-mediated C3 activation. Binding of properdin to C3b could also be blocked by heparin(oids) and recombinant Salp20. This work indicates that properdin serves as a docking station for AP activation on PTECs and a Salp20 analog or heparinoids may be viable inhibitors in properdin mediated AP activation.

Bioprocess strategies for enhancing the outdoor production of Nannochloropsis gaditana: an evaluation of the effects of pH on culture performance in tubular photobioreactors.

A priority of the industrial applications of microalgae is the reduction of production costs while maximizing algae biomass productivity. The purpose of this study was to carry out a comprehensive evaluation of the effects of pH control on the production of Nannochloropsis gaditana in tubular photobioreactors under external conditions while considering the environmental, biological, and operational parameters of the process. Experiments were carried out in 3.0 m3 tubular photobioreactors under outdoor conditions. The pH values evaluated were 6.0, 7.0, 8.0, 9.0, and 10.0, which were controlled by injecting pure CO2 on-demand. The results have shown that the ideal pH for microalgal growth was 8.0, with higher values of biomass productivity (Pb) (0.16 g L-1 d-1), and CO2 use efficiency ([Formula: see text]) (74.6% w w-1); [Formula: see text]/biomass value obtained at this pH (2.42 [Formula: see text] gbiomass-1) was close to the theoretical value, indicating an adequate CO2 supply. At this pH, the system was more stable and required a lower number of CO2 injections than the other treatments. At pH 6.0, there was a decrease in the Pb and [Formula: see text]; cultures at pH 10.0 exhibited a lower Pb and photosynthetic efficiency as well. These results imply that controlling the pH at an optimum value allows higher CO2 conversions in biomass to be achieved and contributes to the reduction in costs of the microalgae production process.

Urinary Properdin and sC5b-9 Are Independently Associated With Increased Risk for Graft Failure in Renal Transplant Recipients.

The pathophysiology of late kidney-allograft failure remains complex and poorly understood. Activation of filtered or locally produced complement may contribute to the progression of renal failure through tubular C5b-9 formation. This study aimed to determine urinary properdin and sC5b-9 excretion and assess their association with long-term outcome in renal transplant recipients (RTR). Methods: We measured urinary properdin and soluble C5b-9 in a well-defined cross-sectional cohort of RTR. Urinary specimens were taken from a morning urine portion, and properdin and sC5b-9 were measured using an enzyme-linked-immunosorbent assay (ELISA). Cox proportional hazard regression analyses were used to investigate prospective associations with death-censored graft failure. Results: We included 639 stable RTR at a median [interquartile range] 5.3 (1.8-12.2) years after transplantation. Urinary properdin and sC5b-9 excretion were detectable in 161 (27%) and 102 (17%) RTR, respectively, with a median properdin level of 27.6 (8.6-68.1) ng/mL and a median sC5b-9 level of 5.1 (2.8-12.8) ng/mL. In multivariable-adjusted Cox regression analyses, including adjustment for proteinuria, urinary properdin (HR, 1.12; 95% CI 1.02-1.28; P = 0.008) and sC5b-9 excretion (HR, 1.34; 95% CI 1.10-1.63; P = 0.003) were associated with an increased risk of graft failure. If both urinary properdin and sC5b-9 were detectable, the risk of graft failure was further increased (HR, 3.12; 95% CI 1.69-5.77; P < 0.001). Conclusions: Our findings point toward a potential role for urinary complement activation in the pathogenesis of chronic allograft failure. Urinary properdin and sC5b-9 might be useful biomarkers for complement activation and chronic kidney allograft deterioration, suggesting a potential role for an alternative pathway blockade in RTR.

Anorexia nervosa and heart disease: a systematic review.

Anorexia nervosa (AN) is an eating disorder that most frequently afflicts females in adolescence. In these subjects, cardiovascular complications are the main cause of morbidity and mortality. Aim of this review is to analyze the hemodynamic, pro-arrhythmic and structural changes occurring during all phases of this illness, including re-feeding. A systematic literature search was performed on studies in the MEDLINE database, from its inception until September 2017, with PUBMED interface focusing on AN and cardiovascular disease. This review demonstrated that the most common cardiac abnormalities in AN are bradycardia and QT interval prolongation, which may occasionally degenerate into ventricular arrhythmias such as Torsades des Pointes or ventricular fibrillation. As these arrhythmias may be the substrate of sudden cardiac death (SCD), they require cardiac monitoring in hospital. In addition, reduced cardiac mass, with smaller volumes and decreased cardiac output, may be found. Furthermore, mitral prolapse and a mild pericardial effusion may occur, the latter due to protein deficiency and low levels of thyroid hormone. In anorectic patients, some cases of hypercholesterolemia may be present; however, conclusive evidence that AN is an atherogenic condition is still lacking, although a few cases of myocardial infarction have been reported. Finally, refeeding syndrome (RFS), which occurs during the first days of refeeding, may engender a critically increased risk of acute, life-threatening cardiac complications.

Severe atypical hand-foot-and-mouth disease in adults due to coxsackievirus A6: Clinical presentation and phylogenesis of CV-A6 strains.

BACKGROUND: Typically, hand-foot-and-mouth disease (HFMD) is a mild childhood illness associated with coxsackievirus (CV)-A16, CV-A6, enterovirus (EV)-A71. OBJECTIVES: To identify the viral agents associated with severe cases of atypical HFMD in Italy. STUDY DESIGN: Epidemiologically unrelated cases of severe atypical HFMD admitted to the Emergency Room (ER) of IRCCS San Martino IST (Genoa, Italy) in 2014-2016 were investigated. Serologic screening for viral positivity was performed against exanthem-inducing agents. Ten cases with serology indicative of recent EV infection were selected. Molecular assays were used to detect viral genomes in blood [EVs, Parvovirus B19 (PVB19), herpesviruses (CMV; EBV, HHV-6, -7, -8)]. RESULTS: CV-A6 was detected in 10 cases of severe atypical HFMD. Two cases were also infected with PVB19. Herpesviruses were not detected. Phylogenetic analysis mapped the CV-A6 strains into a single cluster related to two recent isolates from a German and an Asian child. Fever, systemic symptoms, severe vasculitis-like rash, and enanthem were predominant at presentation. Spontaneous recovery occurred in 1-3 weeks. CONCLUSIONS: CV-A6 is emerging as a frequent cause of severe atypical HFMD in Italian adults. This viral agent is disseminating worldwide. Dermatologists must identify the manifold alterations caused by EVs and understand the diagnostic power of current virology methods.

Fed-batch cultivation with CO2 and monoethanolamine: Influence on Chlorella fusca LEB 111 cultivation, carbon biofixation and biomolecules production.

The aim of this study was to evaluate the interaction between the periodic addition of monoethanolamine (MEA) and CO2 during the cultivation of Chlorella fusca LEB 111. For this purpose, MEA has been added in abiotic assays, followed by fed-batch cultures with that green alga and the absorbent. BG-11 medium shown a higher potential of CO2 absorption with MEA addition, and the bicarbonate was the chemical species of carbon prevailing in the chemical equilibrium. The periodic addition of MEA did not reduce the kinetics of growth, promoted a higher accumulation of DIC (81.4 mg L-1) in the medium and protein (44.0% w w-1) and lipid (30.8% w w-1) concentrations in the biomass of C. fusca LEB 111. Therefore, it was demonstrated that fed-batch culture with MEA increased CO2 fixation and the biomolecule synthesis as proteins and lipids.

Outcome after cholecystectomy in the elderly.

BACKGROUND: Cholecystectomy is considered the standard treatment for acute cholecystitis and symptomatic gallstones. An increasing number of frail elderly patients are being referred for this surgical treatment. A better understanding of surgical outcome in the elderly is needed to improve quality of care. METHODS: A retrospective analysis of 565 patients who underwent cholecystectomy was performed. Focus of the analyses was on postoperative complications and its predictors. RESULTS: The study population was divided in two cohorts; aged <70. More complications were found in patients aged ≥70 years. More elderly patients were admitted to the intensive care, respectively 4.0% and 14.1% (P = 0.045). Hospital mortality was 6% in patients aged ≥70 years vs 0.6% in patients <70. CONCLUSION: In elderly patients, the complication and mortality rate following cholecystectomy is higher than previously reported. For high-risk patients aged ≥70 with cholecystitis, alternative therapies should be considered as a bridge to surgery or definite treatment.

Risk-Assessment of Esophageal Surgery: Diagnosis and Treatment of Celiac Trunk Stenosis.

Anastomotic leakage of the gastric conduit following surgical treatment of esophageal cancer is a life-threatening complication. An important risk factor associated with anastomotic leakage is calcification of the supplying arteries of the gastric conduit. The patency of calcified splanchnic arteries cannot be assessed on routine computed tomography (CT) scans for esophageal cancer and, as such, in selected patients with known or assumed mesenteric artery disease, additional CT angiography of the abdominal arteries with 1 mm slices is strongly encouraged. If the mesenteric perfusion is compromised in patients with resectable esophageal cancer, angioplasty procedures with stenting of the mesenteric arteries could be performed to prevent possible ischemia of the gastric conduit.

Chylopericardium: a case report demonstrating utility of lymphography combined with 3D computed tomography for corrective surgical treatment using VATS.

We present a case of a 58 year-old woman with primary chylopericardium associated with chylothorax. Chylopericardium is a condition in which chylous fluid containing a high concentration of triglycerides accumulates in the pericardial cavity, and it can form for many different reasons. 3D computed tomography with lymphography precisely depicted the specific location of the lymphatic leak in this patient, which was successfully repaired using targeted video assisted thoracic surgery (VATS).

The implantable cardioverter defibrillator in primary prevention: a revision of monocentric study group.

AIMS: To evaluate the outcome of a population implanted with an implantable cardioverter defibrillator (ICD) for primary prevention in terms of mortality, morbidity and appropriate and inappropriate interventions. Secondly, to compare the performances of single-chamber vs. dual-chamber devices. METHODS: We examined all patients with CAD or CMD who received an ICD in primary prevention with at least 6 months of follow-up. For each patient were evaluated, primarily, survival, complications related to the implantation and performance of the device (antitachycardia pacing/shock). RESULTS: Of 193 patients, 163 were men (84.5). Mean age was 64.4 ± 10 years. One hundred and twenty patients (62%) were affected by CAD and 73 (38%) by CMD. The ejection fraction was 26 ± 6%. Fifty-three patients (27.5%) received a dual-chamber ICD, whereas 140 (72.5%) received a single-chamber ICD. There were periprocedural complications in 5.2% of the patients. At a mean follow-up of 49.9 months, 55 patients (28.5%) died. Appropriate interventions were documented in 40 patients (20.7%). In 36 patients (18%), inappropriate interventions occurred. Patients implanted with dual-chamber ICD had an overall mortality of 17% compared to 32.4% for those implanted with single-chamber ICD (P = 0.029). Mortality was higher in patients with CAD (33.9%) (P = 0.032). Among the fatalities, 69% occurred in patients who had an ejection fraction 25% or less at the time of implantation and 31% in patients with an ejection fraction greater than 25% (P = 0.013). CONCLUSIONS: The 4-year survival was 72%. The overall mortality was higher in patients with CAD. More than two-thirds of the deceased had an ejection fraction less than 25%. The dual-chamber ICD patients had a significantly lower mortality rate.

Effect of interleukin-1 gene polymorphism in a periodontally healthy Hispanic population treated with mucogingival surgery.

OBJECTIVES: A genetic test for susceptibility of periodontal disease has been introduced. A positive test indicates a risk factor for more severe periodontal destruction. The prevalence of genotype positive subjects has been reported around 30%. In a Mexican population, we have found a 26% prevalence of genotype positive individuals. Few studies have reported the response to therapy in these individuals. The purpose of this study was to assess the response to mucogingival surgery in an otherwise periodontally healthy Hispanic population. MATERIALS AND METHODS: 22 subjects (7 male and 15 female) with a mean age of 45 years participated. They were treated 3 years prior for the treatment of Types I and II recession defects using connective tissue grafts. No other active periodontal treatment was required, except for preventive maintenance. A full-mouth clinical evaluation was performed which included assessment of gingival inflammation and measurements of probing pocket depth and clinical attachment levels. Mean values per patient were determined. A finger stick blood sample was collected using specially provided DNA filter paper, let dried, and mailed for processing. RESULTS: Results indicated that 5 out of the 22 subjects were genotype positive. The genotype positive subjects presented the following values: GI 1.13+/-0.17, PPD 2.48+/-0.46, and CAL 3.38+/-0.66. The values for the genotype negative subjects were GI 1.06+/-0.14, PPD 2.38+/-0.31 and CAL 3.11+/-0.53. No statistical significant differences were found when both groups were compared (p>0.05). Furthermore, the treatment of the localized recessions was effective and provided similar amount of coverage in genotype positive and negative subjects. However, more genotype negative subjects showed complete coverage of the recession than genotype positive individuals. CONCLUSIONS: Within the limits of this study it is concluded that (1) periodontal health can be maintained with proper preventive maintenance irrespective of the genotype present, (2) the mean response to mucogingival surgery to cover localized gingival recessions is similar irrespective of the IL-1 periodontal genotype, however, full coverage is achieved more frequently in genotype negative subjects.

Coronary aneurysms and stenosis detected with magnetic resonance coronary angiography in a patient with Kawasaki disease.

Coronary artery abnormalities in Kawasaki syndrome develop in about 15-25% of young patients, mostly as aneurysms. In the long-term evolution of coronary artery disease thrombotic occlusion of aneurysms, premature atherosclerosis, and stenosis, are observed. Magnetic resonance is an emerging modality in the angiographic assessment of coronary arteries. The authors report a case of coronary artery aneurysms and stenosis in a 26-year-old patient with Kawasaki disease detected by magnetic resonance coronary angiography and confirmed by conventional coronary angiography.