Pretransplant Single Antigen Bead-Detected HLA Antibodies in Kidney Transplant Long-term Outcome: A Single-Center Cohort Experience.

Single-antigen bead (SAB) platform permits the identification of antibodies not detectable by complement-dependent lymphocytotoxicity test, but their clinical significance is not completely understood. The aim of this study was to evaluate whether the presence of pretransplant SAB-detected antibodies is associated with the development of allograft failure. This is a single-center cohort study with 10-year follow-up in which 573 kidney recipients with negative pretransplant complement-dependent lymphocytotoxicity crossmatch who received transplants at the Kidney Transplant Center of Policlinico, Milan, from deceased donors between 1996 and 2005 were evaluated. Pretransplant plasma samples were retrospectively analyzed by SAB assay. Survival analyses were performed to assess the risk of allograft failures by SAB-detected antibodies. Pretransplant antibodies were found in 160 (28.0%) recipients, of whom 42 subsequently developed an allograft failure for a survival rate of 70.9% (95% confidence interval [CI), 63.5-78.4). Among those without antibodies, 58 (14.0%) returned to dialysis with a survival rate of 84.7% (95% CI, 81.0-88.4). In Cox regression analyses, patients with SAB-positivity had 2-fold higher risk of allograft failure than those who were SAB-negative (hazard ratio, 2.07; 95% CI, 1.39-2.79). Results did not change after adjustment for putative confounders. In conclusion, in this single-center cohort, 10-year allograft survival rate was significantly influenced by the presence of SAB-detected antibodies.

Lung Allocation Score System: First Italian Experience.

BACKGROUND: Lung transplantation is an established therapeutic option for patients with end-stage pulmonary disease. In May 2005, the lung allocation score (LAS) was introduced in the United States to maximize the benefit to the recipient population and reduce waiting list mortality. The LAS has been applied in a region of Italy since March 2016 on a provisional basis. The aims of the study were describing waiting list characteristics and short-term outcomes after lung transplantation before and after LAS introduction. METHODS: All the patients who received transplants between January 1, 2011, and March 15, 2017, were included in our retrospective study. The study population was divided into 2 cohorts (historical cohort and post-LAS cohort) and a comparison among the main perioperative data was performed. RESULTS: The historical cohort consisted of 415 patients on the waiting list with 91 deaths and 199 lung transplants; the post-LAS cohort consisted of 134 patients with 10 deaths on the waiting list and 51 transplants. Median waiting time and mortality on the list decreased from 223 to 106 days (P = .03) and from 11.2% to 7.5% (P > .05), respectively. The transplantation rate increased from 25% to 38% (P = .001) and the probability to receive a transplant in the first year in the post-LAS era increased significantly (P = .004). CONCLUSIONS: The results of the introduction of the LAS system in our region are encouraging and have not shown any adverse short-term effects. The regional coordination decided to prolong the experimental application of LAS in order to accumulate more data and to evaluate medium-term outcomes.

Residual vein obstruction in patients diagnosed with acute isolated distal deep vein thrombosis associated with active cancer.

After acute proximal deep vein thrombosis (DVT) the thrombotic mass decreases, especially during the first months of anticoagulation. The persistence of residual vein obstruction (RVO) may predict future recurrence in patients with cancer-associated DVT. We aimed to evaluate the proportion of patients with RVO after an episode of cancer associated isolated distal DVT (IDDVT), to identify variables associated with RVO, and to provide initial evidence of its association with recurrent VTE. We performed a post-hoc analysis of a multicenter cohort study of patients with isolated cancer-associated acute IDDVT. We included patients who underwent a control ultrasonography at the end of the anticoagulant treatment between day 30 and day 365 after index IDDVT, given that no recurrent VTE had already occurred on anticoagulant treatment. A total of 153 patients had ultrasonographic follow-up after a median of 92 days from index IDDVT: 45.8% had RVO and 54.2% exhibited complete recanalization. Female sex, Body Mass Index > 30 Kg/m2 and involvement of axial calf veins showed the strongest association with RVO. The risk of recurrence was twofold higher in patients with (versus without) RVO. RVO persisted in approximately half of patients with an episode of cancer-associated IDDVT at anticoagulant discontinuation. Patients with RVO appeared to be at a higher risk for recurrent events.

The effect of recanalization on long-term neurological outcome after cerebral venous thrombosis.

Essentials The role of cerebral venous thrombosis (CVT) recanalization on neurologic outcome is still debated. We studied a large cohort of 508 CVT patients with 419 patient years of radiological follow-up. Recanalization rate is high during the first months after CVT and neurologic outcome is favorable. High recanalization grade of CVT independently predicts good neurological outcome. SUMMARY: Background Studies with limited sample size and with discordant results described the recanalization time-course of cerebral venous thrombosis (CVT). The neurological outcome after a first episode of CVT is good, but the role of recanalization on neurological dependence is still debated. Objectives The aim of the study is to assess the recanalization rate after cerebral venous thrombosis (CVT) and its prognostic role in long-term neurological outcome. Patients/Methods In a retrospective observational multicenter cohort study, patients with an acute first episode of CVT with at least one available imaging test during follow-up were enrolled. Patency status of the vessels was categorized as complete, partial or not recanalized. Neurological outcome was defined using the modified Rankin scale (mRS) as good (mRS = 0-1) or poor (mRS = 2-6). Results Five-hundred and eight patients (median [IQR] age, 39 [28.5-49] years; 26% male) were included. Complete or partial recanalization was not differently represented in patients undergoing scans at different periods of time (from 28-day to 3 month-period up to a 1-3 year-period). mRS at the time of follow-up imaging was available in 483 patients; 92.8% of them had a mRS of 0-1. CVT recanalization (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.59-4.13) was positively associated, whereas cancer (OR, 0.29; 95% CI, 0.09-0.88), and personal history of venous thromboembolism (VTE) (OR, 0.36; 95% CI, 0.14-0.92) were negatively associated as independent predictors of favorable (mRS = 0-1) outcome at follow-up. Conclusions Most patients with a first CVT had complete or partial recanalization at follow-up. Recanalization was independently associated with a favorable neurological outcome.

Clinical course of isolated distal deep vein thrombosis in patients with active cancer: a multicenter cohort study.

Essentials Isolated distal deep vein thrombosis (IDDVT) is frequently associated with cancer. No study has specifically evaluated the long-term clinical course of cancer-associated IDDVT. Patients with cancer-associated IDDVT are at very high risk of symptomatic recurrence and death. We observed low rates of major bleeding during anticoagulation. SUMMARY: Background Although isolated distal deep vein thrombosis (IDDVT) is frequently associated with cancer, no study has specifically evaluated the long-term clinical course of IDDVT in this setting. Aim To provide data on the rate of recurrent venous thromboembolism (VTE), major bleeding events and death in IDDVT patients with active cancer. Patients and Methods Consecutive patients with active cancer and an objective IDDVT diagnosis (January 2011 to September 2014) were included from our files. We collected information on baseline characteristics, IDDVT location and extension, VTE risk factors, and type and duration of anticoagulant treatment. Results A total of 308 patients (mean age 66.2 [standard deviation (SD), 13.2 years]; 57.1% female) with symptomatic IDDVT and a solid (n = 261) or hematologic (n = 47) cancer were included at 13 centers. Cancer was metastatic in 148 (48.1%) patients. All but three (99.0%) patients received anticoagulant therapy, which consisted of low-molecular-weight heparin in 288 (93.5%) patients. Vitamin K antagonists were used for the long-term treatment in 46 (14.9%) patients, whereas all others continued the initial parenteral agent for a mean treatment duration of 4.2 months (SD, 4.6 months). During a total follow-up of 355.8 patient-years (mean, 13.9 months), there were 47 recurrent objectively diagnosed VTEs for an incidence rate of 13.2 events per 100 patient-years. During anticoagulant treatment, the annual incidence of major bleeding was 2.0 per 100 patient-years. Conclusions Cancer patients with IDDVT have a high risk of VTE recurrence. Additional studies are warranted to investigate the optimal intensity and duration of anticoagulant treatment for these patients.

Pregnancy outcome after a first episode of cerebral vein thrombosis.

Essentials Little is known about recurrences and pregnancy outcome after cerebral vein thrombosis (CVT). We studied a cohort of pregnant women with CVT. Women with CVT appear at increased risk of late obstetrical complications despite prophylaxis. Risks of recurrent thrombosis and bleeding in women on heparin prophylaxis while pregnant are low. SUMMARY: Background The risk of recurrent thrombosis and bleeding episodes in women with previous cerebral vein thrombosis (CVT) on antithrombotic prophylaxis with low-molecular-weight heparin (LMWH) during pregnancy is not established and little information is available on pregnancy outcome. Objectives The aims of this study were to evaluate the risk of obstetrical complications, recurrent venous thrombosis and bleeding in a cohort of pregnant women on LMWH after a first episode of CVT. In addition, to estimate the relative risk of obstetrical complications, patients were compared with healthy women without thrombosis and with at least one pregnancy in their life. Patients We studied a cohort of 52 patients and 204 healthy women. Results The risk of developing late obstetrical complications was 24% (95% CI, 18-38%), leading to a relative risk of 6.09 (95% CI, 2.46-15.05). The risk of miscarriage was not increased. The higher risk of late obstetrical complications in patients appeared unrelated to a previous history of obstetrical complications, to the carriership of thrombophilia abnormalities, or to the presence of co-morbidities. The incidence of termination observed in patients with thrombophilia was double that observed in those without. No recurrent thrombosis or bleeding episodes were observed. Conclusions Women with previous CVT on LMWH prophylaxis during pregnancy have a low risk of developing recurrent thrombosis or bleeding episodes, but seem to have an increased risk of late obstetrical complications.

OC-01 - Clinical history of cancer patients with isolated distal deep vein thrombosis: a multicenter cohort study.

INTRODUCTION: Isolated distal deep vein thrombosis (IDDVT) accounts for one-fourth to one-half of all deep vein thrombosis (DVT) of the leg. Patients with IDDVT are frequently treated for a shorter period of time compared to patients with proximal DVT and/or pulmonary embolism (PE) due to a perceived lower risk of recurrence. About 10-20% of patients with venous thromboembolic events (VTEs) have concomitant cancer. Guidelines recommend long-term anticoagulant treatment in this group of patients due to their high risk of VTE recurrence. Unfortunately, information on the clinical history of IDDVT patients is limited and, to date, no study has evaluated the long-term risk of VTE recurrence in IDDVT patients with cancer. AIM: To provide information on the clinical history of IDDVT patients with active cancer. MATERIALS AND METHODS: A multicenter, cohort study including active-cancer patients with an objective diagnosis of IDDVT (between January 2011 and September 2014) was conducted. Information on baseline characteristics, thrombosis location and extension, concomitant risk factors, type and duration of treatment was collected. All patients were followed for a minimum of 12 months and up to 24 months. During follow-up, VTE recurrence, major bleeding episodes and death were registered. Potential risk factors for VTE recurrence were evaluated. RESULTS: 308 patients (mean age 66.2±13.2 years, female 57.1%) in 13 centers were included, Table 1; 261 patients had solid cancer and 47 patients hematologic cancer. At the time of IDDVT diagnosis, the disease was metastatic in 148 patients (48.1%); 99.0% of patients received anticoagulant treatment: 288 patients (93.5%) were initially treated with low molecular weight heparin, 15 with fondaparinux (5.2%) and 1 with unfractionated heparin; vitamin K antagonists were used in 46 patients (14.9%) only. Total follow-up was 389 patient-years, mean follow-up 15.2 months. Mean duration of treatment was 4.2 months. During the study period there were 47 episodes of VTE recurrence (36 proximal DVT or PE) for a incidence rate of 13.2 events per 100 patient-years; 7 patients had major bleeding (2.3%) and 137 died (44.5%). At multivariate analysis, previous VTE was associated with an increased risk of recurrence (OR 2.10; 95% 1.06, 4.14), whereas patients with gastrointestinal cancer had a lower risk of recurrence (OR 0.26; 95% CI 0.08, 0.86). CONCLUSIONS: Cancer patients with IDDVT have a high risk of VTE recurrence. Other studies are warranted to address the adequate management of these patients.

Next-generation sequencing study finds an excess of rare, coding single-nucleotide variants of ADAMTS13 in patients with deep vein thrombosis.

BACKGROUND: The considerable genetic predisposition to deep vein thrombosis (DVT) is only partially accounted for by known genetic risk variants. Rare single-nucleotide variants (SNVs) of the coding areas of hemostatic genes may explain part of this missing heritability. The ADAMTS13 and VWF genes encode two interconnected proteins with fundamental hemostatic functions, the disruption of which may result in thrombosis. OBJECTIVES: To study the distribution and burden of rare coding SNVs of ADAMTS13 and VWF found by sequencing in cases and controls of DVT. PATIENTS/METHODS: The protein-coding areas of 186 hemostatic/proinflammatory genes were sequenced by next-generation technology in 94 thrombophilia-negative patients with DVT and 98 controls. Gene-specific information on ADAMTS13 and VWF was used to study the association between DVT and rare coding SNVs of the two genes. RESULTS: More than 70 billion base pairs of raw sequence data were produced to sequence the 700-kb target area with a median redundancy of × 45 in 192 individuals. Most of the 4366 SNVs identified were rare and non-synonymous, indicating pathogenetic potential. Rare (frequency of < 1%) and low-frequency (< 5%) coding SNVs of ADAMTS13 were associated with DVT (prevalence 17% vs. 4%; odds ratio [OR] 4.8 and 95% confidence interval [CI] 1.6-15.0 for rare coding; prevalence 36% vs. 23%, OR 1.9 and 95% CI 1.0-3.5 for low-frequency coding). Patients with rare coding SNVs of ADAMTS13 had lower plasma levels of ADAMTS-13 activity than patients without them. SNVs of VWF were not associated with DVT. CONCLUSIONS: We found an excess of rare coding SNVs of the ADAMTS13 gene in patients with DVT.

Cytotoxicity and uptake of archaeosomes prepared from Aeropyrum pernix lipids.

Archaeon Aeropyrum pernix K1 is an obligate aerobic hyperthermophilic organism with C25,25-archeol membrane lipids with head groups containing inositol. Interactions of archaeosomes, liposomes prepared from lipids of A. pernix, with mammalian cells in vitro were studied. In vitro cytotoxicity was tested on five different cell lines: rodent mouse melanoma cells (B16-F1) and Chinese hamster ovary (CHO) cells, and three human cell lines-epithelial colorectal adenocarcinoma cells (CACO-2), liver hepatocellular carcinoma cell line (Hep G2) and endothelial umbilical vein cell line (EA.hy926). Archaeosomes were nontoxic to human Hep G2, CACO-2 and mildly toxic to rodent CHO and B16-F1 cells but showed strong cytotoxic effect on EA.hy926 cells. Confocal microscopy revealed that archaeosomes are taken up by endocytosis. The uptake of archaeosomes and the release of loaded calcein are more prominent in EA.hy926 cells, which is in line with high toxicity toward these cells. The mechanisms of uptake, release and action in these cells as well as in vivo functioning have to be further studied for possible targeted drug delivery.

The endothelial plasma membrane transporter bilitranslocase mediates rat aortic vasodilation induced by anthocyanins.

BACKGROUND AND AIMS: Anthocyanins, a sub-class of flavonoids, induce endothelium-dependent vasorelaxation, by activating endothelial nitric oxide synthase and consequently increasing production of the vasorelaxant agent nitric oxide. It is not yet clear if anthocyanin-induced vasorelaxation starts with their interaction with plasma membrane receptors in the extracellular compartment, or with their membrane transport toward intracellular molecular targets. We therefore investigated the possible role of bilitranslocase (TC 2.A.65.1.1), an endothelial plasma membrane carrier that transports flavonoids, in the vasodilation activity induced by anthocyanins. METHODS AND RESULTS: Vascular reactivity was assessed in thoracic aortic rings obtained from male Wistar rats. Pre-treatment of aortic rings with anti-sequence bilitranslocase antibodies targeting the carrier, decreased vasodilation induced by cyanidin 3-glucoside and bilberry anthocyanins. CONCLUSION: Here we show for the first time that bilitranslocase mediates a critical step in vasodilation induced by anthocyanins. This offers new insights into the molecular mechanism involved in endothelium-dependent vasorelaxation by flavonoids, and the importance of their specific membrane carriers.

Low borderline plasma levels of antithrombin, protein C and protein S are risk factors for venous thromboembolism.

BACKGROUND: Inherited deficiencies of antithrombin (AT), protein C (PC) and protein S (PS) are risk factors for venous thromboembolism (VTE). They are usually defined by laboratory cut-offs (in our setting 81, 70 and 63 IU dL(-1), respectively), which give only a rough idea of the VTE risk associated with plasma levels of these proteins. OBJECTIVES: We investigated whether the risk of VTE associated with the plasma deficiencies of AT, PC or PS has a dose-response effect, and whether low borderline levels of these proteins are associated with an increased risk of VTE, both in the whole study population and separately in carriers of either factor V Leiden or G20210A prothrombin gene mutation. PATIENTS/METHODS: A case-control study of 1401 patients with a first objectively-documented VTE and 1847 healthy controls has been carried out. RESULTS: A dose-response effect on the VTE risk was observed for all the three anticoagulant proteins. Compared with individuals with AT, PC or PS levels > 100 IU/dL, the adjusted odds ratio (95% CI) of VTE was 2.00 (1.44-2.78) for AT levels between 76 and 85 IUdL(-1) , 2.21 (1.54-3.18) and 1.84 (1.31-2.59) for PC and PS levels between 61 and 75 IUdL(-1) . The risk of unprovoked VTE in factor V Leiden or prothrombin G20210A carriers appears 2 to 3-fold increased when levels of AT or PS are low borderline. CONCLUSIONS: Low borderline plasma levels of AT, PC and PS are associated with a 2-fold increased risk of VTE and should be considered in the assessment of the individual VTE risk.

The JAK2 V617F mutation in patients with cerebral venous thrombosis.

BACKGROUND: It is currently unclear whether or not cerebral venous thrombosis, such as splanchnic venous thrombosis, can be the first manifestation of an underlying myeloproliferative neoplasm. OBJECTIVE: To determine the prevalence of the JAK2 V617F mutation in patients with a first episode of cerebral venous thrombosis. PATIENTS: In this retrospective cohort study, patients with cerebral venous thrombosis were tested for the JAK2 V617F mutation and were followed until the development of a myeloproliferative neoplasm or censored at the end of follow-up. RESULTS: Ten of 152 patients (6.6%) carried the JAK2 V617F mutation. Three of them had known acquired risk factors for thrombosis, and five had thrombophilia. Six patients met the diagnostic criteria for myeloproliferative neoplasm at the time of cerebral venous thrombosis, and three additional patients developed the disease during the follow-up (median duration 7.8 years, range 6 months to 21.3 years), giving an annual incidence of 0.26% patient-years (95% confidence interval 0.05-0.64). The last patient has no evidence of disease after 3 years of follow-up. Patients without the JAK2 V617F mutation at the time of cerebral venous thrombosis were retested at the end of the follow-up and remained negative, with normal blood counts (log-rank test χ(2) : 159 [P<0.0001]). CONCLUSIONS: Cerebral venous thrombosis can be the first symptom of a myeloproliferative neoplasm. Patients with cerebral venous thrombosis can carry the JAK2 V617F mutation, irrespective of blood count.

Effect of prothrombin 19911 A>G polymorphism on the risk of cerebral sinus-venous thrombosis.

BACKGROUND AND PURPOSE: The A>G polymorphism at position 19911 of the prothrombin gene is associated with a mildly increased risk of venous thromboembolism, alone or in association with such common thrombophilia mutations as factor V Leiden and prothrombin 20210 GA. Its role in cerebral sinus-venous thrombosis (CSVT) is not known. METHODS: The presence of prothrombin 19911 A>G was investigated in a case­control study of 107 patients with cerebral thrombosis and factor V Leiden (n = 25), prothrombin 20210 GA (n = 47), without known thrombophilia (n = 35) and 842 healthy individuals with the corresponding coagulation profile. RESULTS: Prothrombin 19911 A>G did not increase the risk of CSVT in carriers of factor V Leiden (adjusted odds ratio 1.6, 95%CI 0.6­4.7), prothrombin 20210 GA (odds ratio 1.1, 95%CI 0.6­2.2), nor in patients without known thrombophilia (odds ratio 1.3, 95%CI 0.5­3.1). CONCLUSIONS: Prothrombin 19911 A>G polymorphism does not appear to be a risk factor for CSVT, alone or in association with factor V Leiden or prothrombin 20210GA.

Evidence for a putative flavonoid translocator similar to mammalian bilitranslocase in grape berries (Vitis vinifera L.) during ripening.

During maturation, Vitis vinifera berries accumulate a large amount of several anthocyanins in the epidermal tissue, whereas their precursors and intermediates are ubiquitously synthesized within the fruit. Up to date, several mechanisms of flavonoid transport at subcellular level have been hypothesized, but it is not possible to identify a general model applicable in every plant tissue and organ. Recently, a putative anthocyanin carrier, homologue to mammalian bilitranslocase (BTL) (TC 2.A.65.1.1), was found in Dianthus caryophyllus petal microsomes. In the present paper, an immunohistochemical and immunochemical analysis, using an antibody raised against a BTL epitope, evidences the expression and function of such a transporter in V. vinifera berries (cv. Merlot). Specific localisations of the putative carrier within berry tissues together with expression changes during different developmental stages are shown. Water stress induces an increase in protein expression in both skin and pulp samples. A bromosulfalein (BSP) uptake activity, inhibitable by the BTL antibody, is detected in berry mesocarp microsomes, with K (m) = 2.39 microM BSP and V (max) = 0.29 micromol BSP min(-1) mg(-1) protein. This BSP uptake is also competitively inhibited by quercetin (K (i) = 4 microM). A putative role for this carrier is discussed in relation to the membrane transport of secondary metabolites.

On the mechanism of bilitranslocase transport inactivation by phenylmethylsulphonyl fluoride.

Bilitranslocase is a plasma membrane carrier involved in the uptake of bilirubin and other organic anions from the blood into the liver cell. In the membrane, the carrier occurs as two interchangeable metastable forms, with high and low affinity for the substrates, respectively. The latter form can be specifically produced by either cysteine- or arginine modification. In liver plasma membrane vesicles, the serine-specific reagent phenylmethylsulphonyl fluoride is a partial inhibitor of bilitranslocase-mediated BSP transport rate. In this work, phenylmethyl-sulphonyl fluoride is shown to reduce the carrier maximal transport rate, without affecting its affinity for that substrate. In addition, it is found that the chemical modification caused by this reagent neither influences the equilibrium between the high- and the low-affinity forms nor prevents their free interconversion. From the effects of combined derivatizations of cysteine(s), arginine(s) and serine(s), it is concluded that the functionally relevant aminoacid residues lie in a close spatial arrangement. Also, in this study, the PMSF-modified serine(s) is shown to be involved in bilirubin binding by bilitranslocase.

Specific sequence-directed anti-bilitranslocase antibodies as a tool to detect potentially bilirubin-binding proteins in different tissues of the rat.

The hypothesis that the uneven distribution of bilirubin in the organism, which occurs in hyperbilirubinemia, could reflect an uneven distribution of bilirubin-binding proteins was tested by searching for peptides containing the bilirubin-binding motif identified in bilitranslocase (Battiston et al., 1998). In the rat, positive proteins bands were found to be present only in the liver, gastric mucosa and central nervous system. The electrophoretic mobilities of the positive compounds in the liver and stomach were identical to that of purified bilitranslocase (38 kDa). In the brain, on the contrary, two peptides were found with molecular masses of 79 and 34 kDa, respectively. Their distribution pattern in the central nervous system was different for each of them.

The bilirubin-binding motif of bilitranslocase and its relation to conserved motifs in ancient biliproteins.

In the primary structure of bilitranslocase, currently under study in our laboratory, an aminoacid motif was identified and found to be conserved in a number of alpha-phycocyanines, ancient biliproteins present in cyanobacteria. To test the possibility that such a motif could be at least part of the binding site for bilirubin, epitope-specific antibodies were raised. The target corresponds to the sequence 65-75 of bilitranslocase and covers the central portion of the motif identified. The antibodies were shown: 1) to inhibit the electrogenic BSP transport by plasmamembrane vesicles; 2) to react with purified bilitranslocase; and 3) to identify only one protein band with electrophoretic mobility identical to bilitranslocase in Western blots of solubilised plasmamembrane vesicles. The presence of either bilirubin or nicotinate during pre-incubation with the antibodies decreases concentration-wise the inhibition kinetics. From these experiments a dissociation constant of 2.2 +/- 0.3 and 11.3 +/- 1.3 nM for bilirubin-bilitranslocase and nicotinate-bilitranslocase complexes were calculated.