Quorum sensing communication between lipid-based artificial cells.

Population behavior based on quorum sensing communication is a key property of living microorganisms. Here, we show quorum sensing behavior in an artificial cell population consisting of giant lipid vesicles loaded with sender-receiver machinery (enzymes and responsive biomolecules). Our system allows the examination of the collective output based on cell density, fuel concentration and proximity, which are important factors controlling natural quorum sensing behavior.

A Phase I/II Clinical Trial to evaluate the efficacy of baricitinib to prevent respiratory insufficiency progression in onco-hematological patients affected with COVID19: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: Baricitinib is supposed to have a double effect on SARS-CoV2 infection. Firstly, it reduces the inflammatory response through the inhibition of the Januse-Kinase signalling transducer and activator of transcription (JAK-STAT) pathway. Moreover, it reduces the receptor mediated viral endocytosis by AP2-associated protein kinase 1 (AAK1) inhibition. We propose the use of baricinitib to prevent the progression of the respiratory insufficiency in SARS-CoV2 pneumonia in onco-haematological patients. In this phase Ib/II study, the primary objective in the safety cohort is to describe the incidence of severe adverse events associated with baricitinib administration. The primary objective of the randomized phase (baricitinib cohort versus standard of care cohort) is to evaluate the number of patients who did not require mechanical oxygen support since start of therapy until day +14 or discharge (whichever it comes first). The secondary objectives of the study (only randomized phase of the study) are represented by the comparison between the two arms of the study in terms of mortality and toxicity at day+30. Moreover, a description of the immunological related changes between the two arms of the study will be reported. TRIAL DESIGN: The trial is a phase I/II study with a safety run-in cohort (phase 1) followed by an open label phase II randomized controlled trial with an experimental arm compared to a standard of care arm. PARTICIPANTS: The study will be performed at the Institut Català d'Oncologia, a tertiary level oncological referral center in the Catalonia region (Spain). The eligibility criteria are: patients > 18 years affected by oncological diseases; ECOG performance status < 2 (Karnofsky score > 60%); a laboratory confirmed infection with SARS-CoV-2 by means of real -time PCR; radiological signs of low respiratory tract disease; absence of organ dysfunction (a total bilirubin within normal institutional limits, AST/ALT≤2.5 X institutional upper limit of normal, alkaline phosphatase ≤2.5 X institutional upper limit of normal, coagulation within normal institutional limits, creatinine clearance >30 mL/min/1.73 m2 for patients with creatinine levels above institutional normal); absence of HIV infection; no active or latent HBV or HCV infection. The exclusion criteria are: patients with oncological diseases who are not candidates to receive any active oncological treatment; hemodynamic instability at time of study enrollment; impossibility to receive oral medication; medical history of recent or active pulmonary embolism or deep venous thrombosis or patients at high-risk of suffering them (surgical intervention, immobilization); multi organ failure, rapid worsening of respiratory function with requirement of fraction of inspired oxygen (FiO2) > 50% or high-flow nasal cannula before initiation of study treatment; uncontrolled intercurrent illness (ongoing or severe active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements); allergy to one or more of study treatments; pregnant or breastfeeding women; positive pregnancy test in a pre-dose examination. Patients should have the ability to understand, and the willingness to sign, a written informed consent document; the willingness to accept randomization to any assigned treatment arm; and must agree not to enroll in another study of an investigational agent prior to completion of Day +28 of study. An electronic Case Report Form in the Research Electronic Data Capture (REDCap) platform will be used to collect the data of the trial. Removal from the study will apply in case of unacceptable adverse event(s), development of an intercurrent illness, condition or procedural complication, which could interfere with the patient's continued participation and voluntary patient withdrawal from study treatment (all patients are free to withdraw from participation in this study at any time, for any reasons, specified or unspecified, and without prejudice). INTERVENTION AND COMPARATOR: Treatment will be administered on an inpatient basis. We will compare the experimental treatment with baricitinib plus the institutional standard of care compared with the standard of care alone. During the phase I, we will define the dose-limiting toxicity of baricitinib and the dose to be used in the phase 2 part of the study. The starting baricitinib dose will be an oral tablet 4 mg-once daily which can be reduced to 2 mg depending on the observed toxicity. The minimum duration of therapy will be 5 days and it can be extended to 7 days. The standard of care will include the following therapies. Antibiotics will be individualized based on clinical suspicion, including the management of febrile neutropenia. Prophylaxis of thromboembolic disease will be administered to all participants. Remdesivir administration will be considered only in patients with severe pneumonia (SatO2 <94%) with less than 7 days of onset of symptoms and with supplemental oxygen requirements but not using high-flow nasal cannula, non-invasive or invasive mechanical ventilation or extracorporeal membrane oxygenation (ECMO). In the randomized phase, tocilizumab or interferon will not be allowed in the experimental arm. Tocilizumab can be used in patients in the standard of care arm at the discretion of the investigator. If it is prescribed it will be used according to the following criteria: patients who, according to his baseline clinical condition, would be an ICU tributary, interstitial pneumonia with severe respiratory failure, patients who are not on mechanical ventilation or ECMO and who are still progressing with corticoid treatment or if they are not candidates for corticosteroids. Mild ARDS (PAFI <300 mmHg) with radiological or blood gases deterioration that meets at least one of the following criteria: CRP >100mg/L D-Dimer >1,000µg/L LDH >400U/L Ferritin >700ng/ml Interleukin 6 ≥40ng/L. The use of tocilizumab is not recommended if there are AST/ALT values greater than 10 times the upper limit of normal, neutrophils <500 cells/mm3, sepsis due to other pathogens other than SARS-CoV-2, presence of comorbidity that can lead to a poor prognosis, complicated diverticulitis or intestinal perforation, ongoing skin infection. The dose will be that recommended by the Spanish Medicine Agency in patients ≥75Kg: 600mg dose whereas in patients <75kg: 400mg dose. Exceptionally, a second infusion can be assessed 12 hours after the first in those patients who experience a worsening of laboratory parameters after a first favourable response. The use of corticosteroids will be recommended in patients who have had symptoms for more than 7 days and who meet all the following criteria: need for oxygen support, non-invasive or invasive mechanical ventilation, acute respiratory failure or rapid deterioration of gas exchange, appearance or worsening of bilateral alveolar-interstitial infiltrates at the radiological level. In case of indication, it is recommended: dexamethasone 6mg/d p.o. or iv for 10 days or methylprednisolone 32mg/d orally or 30mg iv for 10 days or prednisone 40mg day p.o. for 10 days. MAIN OUTCOMES: Phase 1 part: to describe the toxicity profile of baricitinib in COVID19 oncological patients during the 5-7 day treatment period and until day +14 or discharge (whichever it comes first). Phase 2 part: to describe the number of patients in the experimental arm that will not require mechanical oxygen support compared to the standard of care arm until day +14 or discharge (whichever it comes first). RANDOMISATION: For the phase 2 of the study, the allocation ratio will be 1:1. Randomization process will be carried out electronically through the REDcap platform ( https://www.project-redcap.org/ ) BLINDING (MASKING): This is an open label study. No blinding will be performed. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): The first part of the study (safety run-in cohort) will consist in the enrollment of 6 to 12 patients. In this population, we will test the toxicity of the experimental treatment. An incidence of severe adverse events grade 3-4 (graded by Common Terminology Criteria for Adverse Events v.5.0) inferior than 33% will be considered sufficient to follow with the next part of the study. The second part of the study we will perform an interim analysis of efficacy at first 64 assessed patients and a definitive one will analyze 128 assessed patients. Interim and definitive tests will be performed considering in both cases an alpha error of 0.05. We consider for the control arm this rate is expected to be 0.60 and for the experimental arm of 0.80. Considering this data, a superiority test to prove a difference of 0.20 with an overall alpha error of 0.10 and a beta error of 0.2 will be performed. Considering a 5% of dropout rate, it is expected that a total of 136 patients, 68 for each study arm, will be required to complete study accrual. TRIAL STATUS: Version 5.0. 14th October 2020 Recruitment started on the 16th of December 2020. Expected end of recruitment is June 2021. TRIAL REGISTRATION: AEMPs: 20-0356 EudraCT: 2020-001789-12, https://www.clinicaltrialsregister.eu/ctr-search/search (Not publically available as Phase I trial) Clinical trials: BARCOVID19, https://www.clinicaltrials.gov/ (In progress) FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol."

Drug Delivery Nanosystems for the Localized Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme is one of the most prevalent and malignant forms of central nervous system tumors. The treatment of glioblastoma remains a great challenge due to its location in the intracranial space and the presence of the blood⁻brain tumor barrier. There is an urgent need to develop novel therapy approaches for this tumor, to improve the clinical outcomes, and to reduce the rate of recurrence and adverse effects associated with present options. The formulation of therapeutic agents in nanostructures is one of the most promising approaches to treat glioblastoma due to the increased availability at the target site, and the possibility to co-deliver a range of drugs and diagnostic agents. Moreover, the local administration of nanostructures presents significant additional advantages, since it overcomes blood⁻brain barrier penetration issues to reach higher concentrations of therapeutic agents in the tumor area with minimal side effects. In this paper, we aim to review the attempts to develop nanostructures as local drug delivery systems able to deliver multiple agents for both therapeutic and diagnostic functions for the management of glioblastoma.

Control of endemic multidrug-resistant Gram-negative bacteria after removal of sinks and implementing a new water-safe policy in an intensive care unit.

BACKGROUND: Contaminated handwashing sinks have been identified as reservoirs that can facilitate colonization/infection of patients with multidrug-resistant (MDR) Gram-negative bacteria (GNB) in intensive care units (ICUs). AIM: To assess the impact of removing patients' sinks and implementing other water-safe strategies on the annual rates of ICU-acquired MDR-GNB. METHODS: This six-year quasi-experimental study was conducted from January 2011 to December 2016. The intervention was carried out in August 2014 in two adult ICU wards with 12 rooms each. To assess the changes in annual MDR-GNB rates before and after the intervention, we used segmented regression analysis of an interrupted time-series. Crude relative risk (RR) rates were also calculated. FINDINGS: The incidence rates of MDR-GNB were 9.15 and 2.20 per 1000 patient-days in the pre- and post-intervention periods, respectively. This yielded a crude RR of acquiring MDR-GNB of 0.24 (95% confidence interval: 0.17-0.34). A significant change in level was observed between the MDR-GNB rate at the first point of the post-intervention period and the rate predicted by the pre-intervention time trend. CONCLUSION: The implementation of a new water-safe policy, which included the removal of sinks from all patient rooms, successfully improved the control of MDR-GNB spread in an ICU with endemic infection. Our results support the contribution of sink use with the incidence of MDR-GNB in endemic environments.

Lectin-gated and glycan functionalized mesoporous silica nanocontainers for targeting cancer cells overexpressing Lewis X antigen.

Gated mesoporous silica nanoparticles can deliver payload upon the application of a predefined stimulus, and therefore are promising drug delivery systems. Despite their important role, relatively low emphasis has been placed on the design of gating systems that actively target carbohydrate tumor cell membrane receptors. We describe herein a new Lewis X (Lex) antigen-targeted delivery system comprising mesoporous silica nanoparticles (MSNs) loaded with ATTO 430LS dye, functionalized with a Lex derivative (1) and capped with a fucose-specific carbohydrate-binding protein (Aleuria aurantia lectin (AAL)). This design takes advantage of the affinity of AAL for Lex overexpressed receptors in certain cancer cells. In the proximity of the cells, AAL is detached from MSNs to bind Lex, and selectins in the cells bind Lex in the gated MSNs, thereby inducing cargo delivery. Gated MSNs are nontoxic to colon cancer DLD-1 cells, and ATTO 430LS dye delivered correlated with the amount of Lex antigen overexpressed at the DLD-1 cell surface. This is one of the few examples of MSNs using biologically relevant glycans for both capping (via interaction with AAL) and targeting (via interaction with overexpressed Lex at the cell membrane).

Improved survival among ICU-hospitalized patients with community-acquired pneumonia by unidentified organisms: a multicenter case-control study.

A retrospective analysis from prospectively collected data was conducted in intensive care units (ICUs) at 33 hospitals in Europe comparing the trend in ICU survival among adults with severe community-acquired pneumonia (CAP) due to unknown organisms from 2000 to 2015. The secondary objective was to establish whether changes in antibiotic policies were associated with different outcomes. ICU mortality decreased (p = 0.02) from 26.9 % in the first study period (2000-2002) to 15.7 % in the second period (2008-2015). Demographic data and clinical severity at admission were comparable between groups, except for age over 65 years and incidence of cardiomyopathy. Over time, patients received higher rates of combination therapy (94.3 vs. 77.2 %; p < 0.01) and early (<3 h) antibiotic delivery (72.9 vs. 50.3 %; p < 0.01); likewise, the 2008-2015 group was more likely to receive adequate antibiotic prescription [as defined by the Infectious Diseases Society of America/American Thoracic Society (IDSA/ATS) guidelines] than the 2000-2002 group (70.7 vs. 48.2 %; p < 0.01). Multivariate analysis showed an independent association between decreased ICU mortality and early (<3 h) antibiotic administration [odds ratio (OR) 3.48 [1.70-7.15], p < 0.01] or adequate antibiotic prescription according to guidelines (OR 2.22 [1.11-4.43], p = 0.02). In conclusion, our findings suggest that ICU mortality in severe CAP due to unidentified organisms has decreased in the last 15 years. Several changes in management and better compliance with guidelines over time were associated with increased survival.

Genetic engineering strategies to prevent the effects of antibody and complement on xenogeneic chondrocytes.

Advances in animal transgenesis may allow using xenogeneic chondrocytes in tissue-engineering applications for clinical cartilage repair. Porcine cartilage is rejected by humoral and cellular mechanisms that could be overcome by identifying key molecules triggering rejection and developing effective genetic-engineering strategies. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage protects from galactose α1,3-galactose (Gal)-mediated antibody responses. Now, we studied whether expression of a complement inhibitor provides further protection. First, porcine articular chondrocytes (PAC) were isolated from non-transgenic, single and double transgenic pigs expressing HT and moderate levels of human CD59 (hCD59) and their response to human serum was assessed. High recombinant expression of human complement regulatory molecules hCD59 and hDAF was also attained by retroviral transduction of PAC for further analyses. Complement activation on PAC after exposure to 20 % human serum for 24 hours mainly triggered the release of pro-inflammatory cytokines IL-6 and IL-8. Transgenic expression of HT and hCD59 did not suffice to fully counteract this effect. Nevertheless, the combination of blocking anti-Gal antibodies (or C5a) and high hCD59 levels conferred very high protection. On the contrary, high hDAF expression attained the most dramatic reduction in IL-6/IL-8 secretion by a single strategy, but the additional inhibition of anti-Gal antibodies or C5a did not provide further improvement. Notably, we demonstrate that both hCD59 and hDAF inhibit anaphylatoxin release in this setting. In conclusion, our study identifies genetic-engineering approaches to prevent humoral rejection of xenogeneic chondrocytes for use in cartilage repair.

Exploiting natural anti-carbohydrate antibodies for therapeutic purposes.

Natural anti-carbohydrate antibodies (NAbC) are antibodies that target glycans and are continuously produced without apparent external antigen stimulation. Clinically, NAbC are recognized by the adverse reactions to ABO mismatched blood transfusions or organ transplantation and the rejection of xenografts. These clinical effects do not reflect the biological functions of NAbC. However, they launch the possibility of using NAbC for boosting immunity in different clinical settings by means of: 1) expression of glycan antigens in elements that do not hold them to allow the binding and reactivity of existing NAbC; 2) removal of existing NAbC; 3) manipulation of the glycosylation pattern of NAbC.

Predictors of long-term mortality in patients with cirrhosis undergoing cardiac surgery.

AIM: Little is known regarding the long-term outcome in cirrhotic patients undergoing cardiac surgery. The objective of this study was to identify preoperative and postoperative mortality risk factors and to determine the best predictors of long-term outcome. METHODS: Fifty-eight consecutive cirrhotic patients requiring cardiac surgery between January 2004 and January 2009 were prospectively studied at our institution. Seven patients (12%) died. A complete follow-up was performed in the whole survival group until November 2012 (mean 46±28 months). Variables usually measured on admission and during the first 24 h of the postoperative period were evaluated together with cardiac surgery scores (Parsonnet, EuroSCORE), liver scores (Child-Turcotte-Pugh, Model for End-Stage Liver Disease, United Kingdom End-Stage Liver Disease score), and ICU scores (Acute Physiology and Chronic Health Evaluation II and III, Simplified Acute Physiology Score II and III, Sequential Organ Failure Assessment). RESULTS: Twelve patients (23.5%) died during follow-up; six were Child class A and six class B. Comparing survivors vs. non-survivors using univariate analysis, variables associated with better long-term outcome were lower arterial lactate 24 h after admission (1.7±0.4 vs. 2.1±0.7 mmol·L(-1), P=0.03) and higher urine output in the first 24 h (2029±512 vs. 1575±627 mL, P=0.03). The receiver operating characteristic curve showed that the Simplified Acute Physiology Score III score had the best predictive value for long-term outcome (AUC: 77.4±0.76%; sensitivity: 83.3%; specificity: 64.9%, P=0.005). Multivariate analysis identified Simplified Acute Physiology Score III score (P=0.02) and urine output in the first 24 h (P=0.02) as independent factors associated with long-term outcome. Long-term survival was 82.4% for Child A, 47.6% for Child B and 33.3% for Child C (P=0.001). CONCLUSION: Long-term survival in cirrhotic patients requiring cardiac surgery is a more valuable prognostic measure than short-term survival. Urine output in the first 24 h may be a valuable predictor of long-term outcome in these patients. The Simplified Acute Physiology Score III is also useful.

Inhibition of complement component C5 protects porcine chondrocytes from xenogeneic rejection.

OBJECTIVE: Tissue-based xenografts such as cartilage are rejected within weeks by humoral and cellular mechanisms that preclude its clinical application in regenerative medicine. The problem could be overcome by identifying key molecules triggering rejection and the development of genetic-engineering strategies to counteract them. Accordingly, high expression of α1,2-fucosyltransferase (HT) in xenogeneic cartilage reduces the galactose α1,3-galactose (Gal) antigen and delays rejection. Yet, the role of complement activation in this setting is unknown. DESIGN: To determine its contribution, we assessed the effect of inhibiting C5 complement component in α1,3-galactosyltransferase-knockout (Gal KO) mice transplanted with porcine cartilage and studied the effect of human complement on porcine articular chondrocytes (PAC). RESULTS: Treatment with an anti-mouse C5 blocking antibody for 5 weeks enhanced graft survival by reducing cellular rejection. Moreover, PAC were highly resistant to complement-mediated lysis and primarily responded to human complement by releasing IL-6 and IL-8. This occurred even in the absence of anti-Gal antibody and was mediated by both C5a and C5b-9. Indeed, C5a directly triggered IL-6 and IL-8 secretion and up-regulated expression of swine leukocyte antigen I (SLA-I) and adhesion molecules on chondrocytes, all processes that enhance cellular rejection. Finally, the use of anti-human C5/C5a antibodies and/or recombinant expression of human complement regulatory molecule CD59 (hCD59) conferred protection in correspondence with their specific functions. CONCLUSIONS: Our study demonstrates that complement activation contributes to rejection of xenogeneic cartilage and provides valuable information for selecting approaches for complement inhibition.

Determinants of prescription and choice of empirical therapy for hospital-acquired and ventilator-associated pneumonia.

The objectives of this study were to assess the determinants of empirical antibiotic choice, prescription patterns and outcomes in patients with hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP) in Europe. We performed a prospective, observational cohort study in 27 intensive care units (ICUs) from nine European countries. 100 consecutive patients on mechanical ventilation for HAP, on mechanical ventilation>48 h or with VAP were enrolled per ICU. Admission category, sickness severity and Acinetobacter spp. prevalence>10% in pneumonia episodes determined antibiotic empirical choice. Trauma patients were more often prescribed non-anti-Pseudomonas cephalosporins (OR 2.68, 95% CI 1.50-4.78). Surgical patients received less aminoglycosides (OR 0.26, 95% CI 0.14-0.49). A significant correlation (p<0.01) was found between Simplified Acute Physiology Score II score and carbapenem prescription. Basal Acinetobacter spp. prevalence>10% dramatically increased the prescription of carbapenems (OR 3.5, 95% CI 2.0-6.1) and colistin (OR 115.7, 95% CI 6.9-1,930.9). Appropriate empirical antibiotics decreased ICU length of stay by 6 days (26.3±19.8 days versus 32.8±29.4 days; p=0.04). The antibiotics that were prescribed most were carbapenems, piperacillin/tazobactam and quinolones. Median (interquartile range) duration of antibiotic therapy was 9 (6-12) days. Anti-methicillin-resistant Staphylococcus aureus agents were prescribed in 38.4% of VAP episodes. Admission category, sickness severity and basal Acinetobacter prevalence>10% in pneumonia episodes were the major determinants of antibiotic choice at the bedside. Across Europe, carbapenems were the antibiotic most prescribed for HAP/VAP.

Multi-channel receptors based on thiopyrylium functionalised with macrocyclic receptors for the recognition of transition metal cations and anions.

We report herein the synthesis and characterization of a family of ligands containing different cation binding sites covalently connected to a thiopyrylium signalling reporter. The receptors L1-L6 are able to signal the presence of certain metal cations via three different channels; i.e. electrochemically, fluorogenically and chromogenically. An acetonitrile solution of L1-L6 shows a bright blue colour due to a charge-transfer band in the 575-585 nm region. The colour variation in acetonitrile of L1-L6 in the presence of the metal cations Ag+, Cd2+, Cu2+, Fe3+, Hg2+, Ni2+, Pb2+ and Zn2+ has been studied. A selective hypsochromic shift of the blue band was found for the systems L4-Pb2+ and L5-Hg2+. Additionally, L1-L6 are poorly fluorescent but coordination with certain metal cations induces an enhancement of the fluorescence at ca 500 nm. For instance, the presence of Cu2+ and Fe3+ induced a remarkable 42-fold and 45-fold enhancement in the emission intensity of L1 centred at 500 nm, respectively. Also remarkable was the 18-fold enhancement observed for L4 and L5 in the presence of Fe3+ and Cu2+, respectively. The electrochemical behaviour of receptors L1-L6 was studied in acetonitrile using platinum as a working electrode and [Bu4N][BF4] as a supporting electrolyte. This family of receptors showed a one-electron reversible redox process at ca. -0.46 V versus sce attributed to the reduction of the thiopyrylium group. A moderate anodic shift in the presence of certain metal cations was observed. The effect in the UV-visible spectra of acetonitrile solutions of receptor L1-L6 in the presence of anions was also studied. A remarkable bleaching was found in the presence of cyanide.

Unidad de Cuidados Intermedios de Medicina Interna / Intermediate Care Unit of Internal Medicine

No disponible

Bloodstream infection related to catheter connections: a prospective trial of two connection systems.

Bloodstream infections (BSIs) related to central venous catheters (CVCs) and arterial catheters (ACs) are an increasing problem in the management of critically ill patients. Our objective was to assess the efficacy of a needle-free valve connection system (SmartSite), Alaris Medical Systems, San Diego, CA, USA) in the prevention of catheter-related bloodstream infection (CR-BSI). Patients admitted to an intensive care unit were prospectively assigned to have a CVC and AC connected with either a needle-free valve connection system (NFVCS) or a three-way stopcock connection (3WSC). The characteristics of the patients were similar in the two groups. Before manipulation, the NFVCS was disinfected with chlorhexidine digluconate 0.5% alcoholic solution. The 3WSC was not disinfected between use but it was covered with a protection cap. A total of 799 patients requiring the insertion of a multilumen CVC or AC for >48h from 1 April 2002 to 31 December 2003 were included. CR-BSI rates were 4.61 per 1000 days of catheter use in the disinfected NFVCS group and 4.11 per 1000 days of catheter use in the 3WSC group (P=0.59). When CVC-BSIs and AC-BSIs were analysed separately, the rate of CVC-BSI was 4.26 per 1000 days of catheter use in the NFVCS group, compared with 5.27 in the 3WSC group (P=0.4). The incidence rate of AC-BSI was 5.00 per 1000 days of catheter use in the NFVCS group, compared with 2.83 in the 3WSC group (P=0.08). The use of NFVCS does not reduce the incidence of catheter-related bacteraemia. The arterial catheter (AC) is a significant source of infection in critically ill patients.

The serum level of xenoantibodies, and hDAF or alphaGAL expression on pig cells, modulate in vitro the protection given by hDAF to primate complement-mediated damage.

The ability of human complement regulatory molecules to prevent xenograft rejection following pig-to-primate xenotransplantation is limited. We assayed the efficacy of transgenic human decay accelerating factor (hDAF) expressed on porcine cells to inhibit the in vitro complement activity of primate sera. We measured the cytotoxic activity of baboon or human sera against peripheral blood lymphocytes (PBLs) from hDAF or nontransgenic pigs using a flow cytometry complement-mediated cytotoxicity assay (FCCA). We also analyzed the anti-Galalpha1-3Gal (alphaGal) antibody titer of the baboon sera by ELISA and the expression of hDAF and alphaGal on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. However, the hDAF molecule was more efficient against human than baboon sera. The humoral cytotoxicity capacity correlated with the level of both IgG and IgM anti-alphaGal antibodies. In addition, inhibition of complement-mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and alphaGal molecules on target cells. These results confirm in vitro the protective role of hDAF in pig cells to heterologus complement mediated damage, but they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, low expression of hDAF, or high expression of alphaGal on pig cells.

Liver transplantation from maastricht category 2 non-heart-beating donors: a source to increase the donor pool?

INTRODUCTION: The demand for liver transplantation has increasingly exceeded the supply of cadaver donor organs. Non-heart-beating donors (NHBDs) may be an alternative to increase the cadaver donor pool. The outcome of 20 liver transplants from Maastricht category 2 NHBD was compared with that of 40 liver transplants from heart-beating donors (HBDs). After unsuccessful cardiopulmonary resuscitation (CPR), cardiopulmonary support with simultaneous application of chest and abdominal compression (CPS; n = 6) or cardiopulmonary bypass (CPB; n = 14) was used to maintain the donors. RESULTS: At a minimum follow-up of 2 years, actuarial patient and graft survival rates with livers from Maastricht category 2 NHBD were 80% and 55%, respectively. Transplantation of organs from these donors was associated with a significantly higher incidence of primary nonfunction, biliary complications, and more severe initial liver dysfunction compared with organs from HBDs. The graft survival rates was 83% for livers from NHBDs preserved with CPS and 42% in those maintained with CPB.

Porcine endothelial cell activation in hDAF pig hearts transplanted in baboons with prolonged survival and lack of rejection.

Depletion of anti-alphaGal antibodies before and after transplantation with GAS 914, a polylysine containing alphaGal epitopes, together with immunosuppression, has been shown to prevent acute humoral xenograft rejection (AHXR) in hDAF pig-to-baboon xenotransplantation. This therapy was associated with low levels of serum anti-alphaGal antibodies and lack of antipig hemolytic antibodies (APA) during the entire transplant course. In the present study we investigated the condition of xenograft endothelial cells and the presence of other antipig antibodies. No xenograft failed because of AHXR. However, endothelial cell markers of activation, such as CD62, CD106, ET-1, and particularly 5A6/8, were detected at necropsy, along with a lack or scarce deposits of IgM and total absence of complement and fibrin. The endothelial cell markers were negative or slightly positive at biopsy obtained 30 minutes after transplantation. At the time of animal death serum xenoantibodies against pig aortic cells were also detected by immunochemistry whereas anti-alphaGal and APHA were almost absent, suggesting that the presence of non-anti-alphaGal and noncytotoxic xenoantibodies may cause endothelial activation.

Monitoring cytotoxicity against pig cells after transplantation using two-color fluorescence viability assay.

Acute humoral xenograft rejection (AHXR) is now the major hurdle for the long-term survival of pig organs transplanted into nonhuman primates. Mechanisms involved in this rejection are not well understood, albeit that it has been proposed to require the participation of antibodies with specificities other than alphaGal. In this study, we evaluated a two-color fluorescence method, fluorescein dicetate (FAD)/propodium iodide (PI), to stain live versus dead cells, respectively, to monitor complement-mediated antibody cytotoxicity in hDAF pig-to-baboon xenotrasplants. FDA/PI flow cytometry assays showed a high correlation (rho Spearman=.736; P=.003) with the cytotoxic activities of baboon serum antibodies against PK15 cells, using either endogenous or exogenous complement. Average serum cytotoxicity against AOC40 was higher (59.82+/-17.90) compared with PK15 (33.69+/-13.05) and L35 (37.64+/-12.77) cells, albeit the difference did not reach statistical significance. Incubation of serum samples with low-molecular weight heparin reduced serum cytotoxicity against PK15 cells in dose-dependent fashion. Therefore, FDA-PI two-color fluorescence is a suitable method to study antibody-mediated cytotoxicity by endogenous or exogenous complement for various pig cells.

Elicited non-anti-alphaGAL antibodies may cause acute humoral rejection of hDAF pig organs transplanted in baboons.

The combination of immunosuppression and GAS 914, a polylysine containing alphaGal trisaccharide type 2 (TRI 2), has been associated with the prevention of acute humoral xenograft rejection (AHXR) in human decay accelerating factor (hDAF) pig-to-baboon xenotransplants. The aim of this study was to investigate the role of immunosuppression and GAS 914 to neutralize xenoantibodies before and after xenotransplantation. Eight baboons underwent heteropic heart xenotransplantation with hDAF transgenic pig organs, receiving GAS 914 before and after transplantation. Six baboons (Group A) were treated with an immunosuppression protocol that included cyclophosphamide (CyP), Neoral, ERL, and steroids. The other 2 baboons (Group B) were treated with the same immunosuppression but with a 50% reduction in the doses of CyP. No xenograft from Group A underwent acute humoral xenograft (median survival, 27 days), whereas the 2 in Group B experienced rejection (median survival, 6 days). GAS 914 depleted both immunoglobulin (Ig)M and IgG anti-alphaGAL disaccharide (DI), trisaccharide type 2 (TRI 2), and trisaccharide type 6 (TRI 6), before and after transplantation in Groups A and B. However, cytotoxic antibodies with other anti-pig specificities were elicited by the xenografts in Group B leading to AHXR.