Dual lumen cannulation and mobilization of patients with venovenous extracorporeal membrane oxygenation.

BACKGROUND: Mobilization is important in longer courses in intensive care unit (ICU), typical for patients requiring venovenous extracorporeal membrane oxygenation (V-V ECMO). For patients supported with ECMO, especially out-of-bed mobilizations improve outcome. We hypothesized that utilization of a dual lumen cannula (DLC) for V-V ECMO would facilitate out-of-bed mobilization compared to single lumen cannulas (SLC). METHODS: Retrospective single center registry study including all V-V ECMO patients cannulated between 10/2010 and 05/2021 for respiratory failure. RESULTS: The registry included 355 V-V ECMO patients (median age 55.6 years, 31.8% female, 27.3% with preexisting pulmonary disease), 289/355 (81.4%) primary cannulated with DLC, and 66/355 (18.6%) using SLC. Both groups had similar pre-ECMO characteristics. The runtime of the first ECMO cannula was significantly longer in DLC compared to SLC (169 vs. 115 h, p = 0.015). The frequency of prone positioning during V-V ECMO was similar in both groups (38.4 vs. 34.8%, p = 0.673). There was no difference in in-bed mobilization (41.2 vs. 36.4%, for DLC and SLC, respectively, p = 0.491). Patients with DLC were more often mobilized out-of-bed (25.6 vs. 12.1%, OR 2.495 [95% CI 1.150 to 5.268], for DLC and SLC, respectively, p = 0.023). Hospital survival was similar in both groups (46.4 vs. 39.4%, for DLC and SLC, respectively, p = 0.339). CONCLUSION: Patients cannulated with a dual lumen cannula for V-V ECMO support were significantly more often mobilized out-of-bed. Since mobilization is important in prolonged ICU courses typical for ECMO patients, this might be an important benefit. Other benefits of DLC were the longer runtime of the initial cannula set and fewer suction events.

Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis.

Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.

Corrigendum: Survival outcomes and mobilization during venovenous extracorporeal membrane oxygenation: a retrospective cohort study.

[This corrects the article DOI: 10.3389/fmed.2023.1271540.].

Survival outcomes and mobilization during venovenous extracorporeal membrane oxygenation: a retrospective cohort study.

Introduction: Venovenous extracorporeal membrane oxygenation (V-V ECMO) can be considered in critically ill patient in severe pulmonary failure. However, the mobilization of patients on V-V ECMO can be challenging due to logistic and safety concerns. This study aimed to investigate whether 30 days survival was improved in patients who were mobilized during V-V ECMO support. Methods: We conducted a retrospective cohort all-comer study that included all patients cannulated for V-V ECMO at a single center. Patients with a V-V ECMO duration below 24 h were excluded from the analysis. The patients were grouped based on the ICU mobility scale documented during V-V ECMO support. The primary endpoint was 30 days survival, and secondary endpoints included weaning from ECMO and mechanical ventilation, as well as hospital survival. Results: A total of 343 patients were included in the study, with a median age of 56 years and 32% were female. Among them, 28% had chronic lung disease. The ICU mobilization scale ≥2 during ECMO was documented in 62/343 (18%) patients. There were no significant differences in age, gender and preexisting lung disease. Duration of ICU stay (13.1 vs. 15.6 days), time on ECMO (186 vs. 190 h) and mechanical ventilation (11.2 vs. 13.6 days) were slightly shorter in patients with ICU mobility scale <2 compared to those with ≥2 (all p = 0.0001). However, patients with ICU mobilization scale ≥2 showed significantly better 30 days survival (71.0 vs. 48.0%, OR 2.6 (1.5 to 4.8), p = 0.0012) compared to those with <2. In the ≥2 mobility scale group, a significantly higher number of patients were successfully weaned from the ventilator (61.3 vs. 46.6%, OR 1.8 (1.0 to 3.2), p = 0.049). A stronger correlation was observed between more intense mobilizations, such as being in a standing position (OR 5.0 (1.7 to 14.0), p = 0.0038), and higher 30 days survival. Conclusion: The findings of this study suggest that active mobilization during V-V ECMO support is associated with improved 30 days survival and successful weaning from the respirator. Incorporating mobilization as part of the therapeutic approach during ECMO support may offer potential benefits for critically ill patients.

Interpreting the Benefit and Risk Data in Between-Drug Comparisons: Illustration of the Challenges Using the Example of Mefenamic Acid versus Ibuprofen.

Evidence-based pain therapy should rely on precisely defined and personalized criteria. This includes balancing the benefits and risks not only of single drugs but often requires complex between-drug comparisons. Non-steroidal anti-inflammatory drugs (NSAIDs) have been available for several decades and their use is described in an abundance of guidelines. Most of these guidelines recommend that 'the selection of a particular NSAID should be based on the benefit-risk balance for each patient'. However, head-to-head studies are often lacking or of poor quality, reflecting the lower standards for clinical research and regulatory approval at the time. The inconsistency of approved indications between countries due to national applications adds to the complexity. Finally, a fading research interest once drugs become generic points to a general deficit in the post-marketing evaluation of medicines. Far from claiming completeness, this narrative review aimed to illustrate the challenges that physicians encounter when trying to balance benefits and risks in a situation of incomplete and inconsistent data on longstanding treatment concepts. Ibuprofen and mefenamic acid, the most frequently sold NSAIDs in Austria, serve as examples. The illustrated principles are, however, not specific to these drugs and are generalizable to any comparison of older drugs in daily clinical practice.

2-DE and MALDI-TOF-MS analysis of therapeutic fusion protein abatacept.

2-DE and MALDI-TOF MS are useful techniques for the quality evaluation of medicinal products derived from recombinant DNA technology. The principal objective of this study has been to evaluate the suitability of 2-DE in combination with MALDI-TOF MS for the quality study of the therapeutic recombinant protein, abatacept. 1-DE SDS-PAGE, under reducing and nonreducing conditions, and 2-DE analysis were used for the assessment of M(r) , pI, and enzymatic deglycosylation efficiency of abatacept. 2-DE allowed the assessment of product identity, purity, charge heterogeneity, isoform pattern, and post-translational modifications. Furthermore, optimization of the deglycosylation procedure, charge heterogeneity, and sample preparation for the subsequent MALDI-TOF MS analysis has been addressed. PMF analysis allowed rapid identity confirmation of abatacept.

Dysfunction of the Blood-Brain Barrier-A Key Step in Neurodegeneration and Dementia.

The vascular endothelium in the brain is an essential part of the blood-brain-barrier (BBB) because of its very tight structure to secure a functional and molecular separation of the brain from the rest of the body and to protect neurons from pathogens and toxins. Impaired transport of metabolites across the BBB due to its increasing dysfunction affects brain health and cognitive functioning, thus providing a starting point of neurodegenerative diseases. The term "cerebral metabolic syndrome" is proposed to highlight the importance of lifestyle factors in neurodegeneration and to describe the impact of increasing BBB dysfunction on neurodegeneration and dementia, especially in elderly patients. If untreated, the cerebral metabolic syndrome may evolve into dementia. Due to the high energy demand of the brain, impaired glucose transport across the BBB via glucose transporters as GLUT1 renders the brain increasingly susceptible to neurodegeneration. Apoptotic processes are further supported by the lack of essential metabolites of the phosphocholine synthesis. In Alzheimer's disease (AD), inflammatory and infectious processes at the BBB increase the dysfunction and might be pace-making events. At this point, the potentially highly relevant role of the thrombocytic amyloid precursor protein (APP) in endothelial inflammation of the BBB is discussed. Chronic inflammatory processes of the BBB transmitted to an increasing number of brain areas might cause a lasting build-up of spreading, pore-forming ß-amyloid fragments explaining the dramatic progression of the disease. In the view of the essential requirement of an early diagnosis to investigate and implement causal therapeutic strategies against dementia, brain imaging methods are of great importance. Therefore, status and opportunities in the field of diagnostic imaging of the living human brain will be portrayed, comprising diverse techniques such as positron emissions tomography (PET) and functional magnetic resonance imaging (fMRI) to uncover the patterns of atrophy, protein deposits, hypometabolism, and molecular as well as functional alterations in AD.

Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data.

Image analysis of two-dimensional gels is a crucial step in a proteomic workflow and has a direct impact on obtained qualitative and quantitative data. Since the analysis is a complex process and creates large data amounts, the use of a respective software is inevitable. There are only a few papers published addressing the issue of analysis-based variance; therefore, our aim was to highlight the discrepancy of received results when different commercially available image-tools are used for gel analysis especially in terms of comparability of the obtained outcome when the same digital image set is used. A set of six gels (three replicates per group) of real-life samples was created and examined with two different versions of PD-Quest (Bio-Rad) (version 6.1 and its update version 8.0) and with an external image-tool Delta 2D (Decodon) (version 3.6). Replicate groups were analyzed and compared with each other with regard to volume ratios of a group of significantly changed spots. The study points out significant variations among results depending on the software package used, underlining the importance of a careful investigation of post-experimental processes to receive comparable and reliable results.

Increasing chiral recognition in acetal formation.

The design of substituted lactols is described, which in their reaction with racemic alkyl aryl carbinols react preferentially with one of the enantiomers exhibiting selectivities up to 14:1.

Expression of Claudin-1, Claudin-3 and Claudin-5 in human blood-brain barrier mimicking cell line ECV304 is inducible by glioma-conditioned media.

Up to now no standard cell culture model of the blood-brain barrier is available. However, several models based on primary cells or continuous cell lines have been characterized and described in respect of different applications. One of the most important characteristics of the blood-brain barrier is the restriction of paracellular transport, respectively its tightness. Human cell line ECV304 is one of the promising continuous cell lines for blood-brain barrier modelling due to two reasons: on the one hand the cells are able to form significant tighter layers than most of the other cell lines used and on the other hand several properties of the blood-brain barrier are inducible by using glioma-conditioned medium. Claudins are transmembranal proteins which form the backbone of the tight junctions at the blood-brain barrier. We have investigated the presence and inducibility of the expression of Claudin-1, Claudin-3 and Claudin-5 using immunofluorescence microscopy. For the first time this study proves the presence of Claudin-1, Claudin-3 and Claudin-5 in ECV304 (obtained from ECACC) cell layers and the inducibility of their expression by glioma-conditioned media.

Integrating systems approaches into pharmaceutical sciences.

During the first week of December 2007, the European Federation for Pharmaceutical Sciences (EUFEPS) and BioSim, the major European Network of Excellence on Systems Biology, held a challenging conference on the use of mathematical models in the drug development process. More precisely, the purpose of the conference was to promote the 'Integration of Systems Approaches into Pharmaceutical Sciences' in view of optimising the development of new effective drugs. And a challenge this is, considering both the high attrition rates in the pharmaceutical industry and the failure of finding definitive drug solutions for many of the diseases that plague mankind today. The conference was co-sponsored by the American College of Clinical Pharmacology, the European Center for Pharmaceutical Medicine, and the Swiss Society of Pharmaceutical Sciences and, besides representatives from the European Regulatory Agencies and FDA, the meeting was attended by 75 industrial and some 45 academic participants.

New polyamine-sensitive inhibitors of the NMDA receptor: syntheses and pharmacological evaluation.

Derivatives of 5-(4-aminobutyl)-2-thiophene-octylamine, a potent polyamine-sensitive inhibitor of the NMDA receptor, were synthesized and evaluated as inhibitors of [(3)H]MK-801 binding to rat brain membranes. Alkylations of the terminal amino groups reduced inhibitory potency; only incorporation of the amino group of the short 4-aminobutyl arm into a piperidine ring was tolerated. Substitution of the thiophene nucleus with methyl or ethyl, and its replacement by a benzene nucleus, was of minor influence. The corresponding diguanidines exhibited high potency independent of chain length, whereas their sensitivity to spermine was sharply dependent on chain length. Insertion of an amide bond into the long octylamine arm increased sensitivity to spermine and to Tris buffer. Our results indicate that spermine sensitivity of [(3)H]MK-801 binding inhibition is responsive to subtle changes in inhibitor structure and represents a promising target for pharmaceutical research.

Oligonucleotide charge reversal: 2'-O-lysylaminohexyl modified oligonucleotides.

A novel cationic building nucleoside building block designed for antisense and siRNA oligonucleotides is presented. Protected L-lysine was coupled to 2'-O-aminohexyluridine and the resulting nucleoside was phosphitylated for automated oligonucleotide synthesis. An increasing number of these 2'-O-lysylaminohexyl nucleosides lowered the melting temperature of desoxy-thymidine homododecamers, but the decrease was lower than that for DNA/RNA hybrids. Incubation with an exonuclease showed the exceptionally high resistance against enzymatic degradation. CD spectrometry revealed a gradual transition towards an A-type oligonucleotide structure. Based on these data, the cationic building block is particularly suited for gapmer antisense as well as siRNA oligonucleotides.

Synthesis of thieno[2,3-b]pyridinones acting as cytoprotectants and as inhibitors of [3H]glycine binding to the N-methyl-D-aspartate (NMDA) receptor.

The standard glycine site antagonist of the N-methyl-D-aspartate (NMDA) receptor, 3-phenyl-4-hydroxyquinolin-2(1H)-one (21), was used as a template for bioisostere benzene/thiophene exchange. Phenylacetylation of aminothiophene carboxylic acid methyl esters and subsequent cyclization delivered the three possible thienopyridinone isomers. 4-Hydroxy-5-phenylthieno[2,3-b]pyridin-6(7H)-one (3a), with the shortest distance between the sulfur and the nitrogen atom, was the most potent isomer (K(i) against the binding of [(3)H]glycine to rat membranes 16 microM), comparable in potency to the model quinolinone (21, 12 microM). Replacement of the phenyl substituent of 21 by a 2-thienyl residue resulted in a 2-5-fold loss in potency and was abandoned. In the thieno part of the thienopyridinone nucleus, the most successful substituents were halogen (Cl or Br) close to the sulfur atom and short alkyl chains at the other position, resulting in 7h, 8h, 8i, and 8m, with K(i) values between 5.8 and 10.5 nM. Introduction of a 3'-phenoxy moiety yielded several compounds with still higher potencies (18h, 18i, 18l, and 18m; K(i) between 1.1 and 2.0 nM). Quantitative structure-activity relationship (QSAR) calculations resulted in a consistent interpretation of the potencies of most compounds. Several of these 3'-phenoxy derivatives protected mouse fibroblast cell lines with transfected NMDA receptors from glutamate-induced toxicity. In addition, we report in vivo results for four of these compounds.

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2.

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.

APTS-labeled dextran ladder: a novel tool to characterize cell layer tightness.

The aim of this work was the development of an easy manageable analytic system for describing tightness of cell layers in a molecular size dependent manner, which is more precise than currently used ones. Dextrans were labeled by reductive amination with fluorescent 1-aminopyrene-3,6,8-trisulfonate (APTS). This mixture, including internal standard diazepam, was used for transport studies, which were accomplished with an established transwell blood-brain barrier model culturing an immortalized porcine brain microvascular endothelial cell line (PBMEC/C1-2). Samples were analyzed by fluorescence measurements, capillary electrophoresis and RP-LC. Following this approach, a permeability pattern could be achieved including each single fraction from APTS, APTS-glucose to APTS-dextran consisting of 31 glucose units. Permeability coefficients were calculated and ranged from 16.38+/-3.79 microm/min for APTS to 6.07+/-1.23 microm/min for the APTS-dextran with 31 glucose units (diazepam: 67.97+/-7.32 microm/min). All in all, the developed APTS-dextran ladder is an useful tool to characterize cell layer tightness--especially to describe paracellular transport ways and leakiness status of the blood-brain barrier over time--applying a wide range from smaller to larger molecules at the same time in order to refine, e.g. TEER, sucrose or Evans blue measurements.

A novel concept for ligand attachment to oligonucleotides via a 2'-succinyl linker.

Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects. In this report, a new method for synthesizing oligonucleotide conjugates is described. 2'-Amino-2'-deoxy-5'-dimethoxytrityl-uridine was select ively acylated with a succinic acid linker at the 2' position. This compound was incorporated at the 3' end of an oligonucleotide corresponding to the sequence of Oblimersen. The carboxyl group was protected for oligonucleotide synthesis as a benzyl ester, which could be selectively cleaved at the solid phase by a catalytic phase transfer reaction using palladium nanoparticles as catalyst. An oligonucleotide-fluorescein conjugate was prepared by condensation of aminofluorescein. Circular dichroism spectroscopic experiments showed a B-DNA type structure. The melting temperature of the duplex was only slightly lower than that of Oblimersen. Biological activity measured by western blotting resulted in a Bcl-2 target downregulation nearly identical to that of control Oblimersen on human melanoma cells, proving that this method is attractive for the binding of ligands located in the minor groove.

Diosgenin contents and DNA fingerprint screening of various yam (Dioscorea sp.) genotypes.

In addition to the importance of many Dioscorea species (yams) as starchy staple food, some representatives are known and still used as a source for the steroidal sapogenin diosgenin, which, besides phytosterols derived from tall-oil, is an important precursor for partial synthesis of steroids for pharmaceutical research and applications. While in edible yams the diosgenin content should be as low as possible, a high yield of the compound is preferable for cultivars which are grown for the extraction of sterols. In the past, miscalculations and insufficiently precise techniques for quantification of diosgenin prevailed. Therefore we set out to re-evaluate the steroid content of a world collection of Dioscorea species, using leaves as sample material. We optimized diosgenin quantification techniques and fingerprinted the whole collection with the DNA amplification fingerprinting (DAF) technique. Total diosgenin contents ranged from 0.04 to 0.93% of dry weight within the collection. Several Dioscorea cultivars can be characterized via their DAF fingerprint patterns.

Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application.