A robust computational quest: Discovering potential hits to improve the treatment of pyrazinamide-resistant Mycobacterium tuberculosis.

The rise of pyrazinamide (PZA)-resistant strains of Mycobacterium tuberculosis (MTB) poses a major challenge to conventional tuberculosis (TB) treatments. PZA, a cornerstone of TB therapy, must be activated by the mycobacterial enzyme pyrazinamidase (PZase) to convert its active form, pyrazinoic acid, which targets the ribosomal protein S1. Resistance, often associated with mutations in the RpsA protein, complicates treatment and highlights a critical gap in the understanding of structural dynamics and mechanisms of resistance, particularly in the context of the G97D mutation. This study utilizes a novel integration of computational techniques, including multiscale biomolecular and molecular dynamics simulations, physicochemical and medicinal chemistry predictions, quantum computations and virtual screening from the ZINC and Chembridge databases, to elucidate the resistance mechanism and identify lead compounds that have the potential to improve treatment outcomes for PZA-resistant MTB, namely ZINC15913786, ZINC20735155, Chem10269711, Chem10279789 and Chem10295790. These computational methods offer a cost-effective, rapid alternative to traditional drug trials by bypassing the need for organic subjects while providing highly accurate insight into the binding sites and efficacy of new drug candidates. The need for rapid and appropriate drug development emphasizes the need for robust computational analysis to justify further validation through in vitro and in vivo experiments.

Eye drops of human origin-Current status and future needs: Report on the workshop organized by the ISBT Working Party for Cellular Therapies.

BACKGROUND AND OBJECTIVES: Serum eye drops (SEDs) are used to treat ocular surface disease (OSD) and to promote ocular surface renewal. However, their use and production are not standardized, and several new forms of human eye drops have been developed. MATERIALS AND METHODS: The International Society for Blood Transfusion Working Party (ISBT WP) for Cellular Therapies held a workshop to review the current types of eye drops of human origin (EDHO) status and provide guidance. RESULTS: The ISBT WP for Cellular Therapies introduced the new terminology 'EDHO' to emphasize that these products are analogous to 'medical products of human origin'. This concept encompasses their source (serum, platelet lysate, and cord blood) and the increasingly diverse spectrum of clinical usage in ophthalmology and the need for traceability. The workshop identified the wide variability in EDHO manufacturing, lack of harmonized quality and production standards, distribution issues, reimbursement schemes and regulations. EDHO use and efficacy is established for the treatment of OSD, especially for those refractory to conventional treatments. CONCLUSION: Production and distribution of single-donor donations are cumbersome and complex. The workshop participants agreed that allogeneic EDHO have advantages over autologous EDHO although more data on clinical efficacy and safety are needed. Allogeneic EDHOs enable more efficient production and, when pooled, can provide enhanced standardization for clinical consistency, provided optimal margin of virus safety is ensured. Newer products, including platelet-lysate- and cord-blood-derived EDHO, show promise and benefits over SED, but their safety and efficacy are yet to be fully established. This workshop highlighted the need for harmonization of EDHO standards and guidelines.

STED lithography in microfluidics for 3D thrombocyte aggregation testing.

Three-dimensional photopolymerization techniques such as multiphoton polymerization lithography (MPL) and stimulated emission depletion (STED) lithography are powerful tools for fabricating structures in the sub-µm range. Combining these techniques with microfluidics enables us to broaden the range of their applications. In this study, we show a microfluidic device enhanced with MPL structures carrying STED-lithographically written nanoanchors that promote binding of the von Willebrand factor (vWF). The density of vWF is adjusted by varying the number of the nanoanchors on the 3D structures. This allows us to study the impact of the density of vWF on the activation of thrombocytes. The activation of the thrombocytes seems to decrease with the density of vWF on the 3D scaffolds inside the microfluidic channels.

Opioid Prescribing After Discharge in a Previously Mechanically Ventilated, Opioid-Naïve Cohort.

BACKGROUND: Opioids are utilized for pain management during and after mechanical ventilation in the intensive care unit (ICU). OBJECTIVE: The purpose of this study was to determine the percentage of potentially unnecessary opioid prescriptions on discharge in previously opioid-naïve patients. METHODS: This retrospective cohort study included mechanically ventilated, opioid-naïve ICU patients who received opioids. The primary outcome of this study was the discrepancy between the amounts of opioids prescribed at discharge versus those likely required based on actual 24-hour prehospital discharge opioid requirements. RESULTS: A total of 71 patients were included. Of these, 63.3% (n = 45) of discharge prescriptions were in alignment with 24-hour predischarge requirements, and 36.7% (n = 26) of discharge prescriptions were in excess of calculated predischarge requirements. At discharge, 57.7% (n = 41) of patients received a nonopioid analgesic. Multivariable linear regression revealed that cardiothoracic ICU admission was associated with an increased risk of inappropriate discharge opioid prescribing, whereas a shorter duration of inpatient oral opioid therapy decreased risk of inappropriate discharge prescribing. CONCLUSION AND RELEVANCE: Opioid prescribing for previously mechanically ventilated patients warrants improvement as a part of the discharge planning process. Application of these data may aid in the reduction of opioid overprescribing at discharge after an ICU stay.

Comparison of fresh frozen plasma vs. coagulation factor concentrates for reconstitution of blood: An in vitro study.

BACKGROUND: Many trauma centres have adopted the administration of fixed ratios of packed red blood cells (PRBCs), platelet concentrates and fresh frozen plasma (FFP) for bleeding patients. However, the haemostatic efficacy of this concept is not well proven. OBJECTIVE: Our objective was to characterise the haemostatic profile of different ratios (2 : 1 : 1, 1 : 1 : 1 and 1 : 1 : 2) of PRBCs, platelet concentrates and FFP in comparison with coagulation factor concentrates (fibrinogen and/or prothrombin complex concentrate). DESIGN: An in vitro study. SETTING: Research laboratories of the department of transfusion medicine, Linz, Austria. MATERIALS: Whole blood donations from a total of 20 male volunteers. INTERVENTION: Reconstitution of blood at different ratios of PRBCs, platelet concentrates and FFP or coagulation factor concentrates. MAIN OUTCOME MEASURES: Cell count, conventional and thromboelastometric coagulation parameters, single coagulation factor activities as well as endogenous thrombin potential. RESULTS: Fibrinogen levels and haematocrit were lower in the FFP group at any ratio compared with the concentrate-based groups (P < 0.0001). Reconstitution of blood with FFP at different ratios resulted in haematocrit or fibrinogen levels that were borderline with regard to recommended substitution triggers (haematocrit 41 ±â€Š2% and fibrinogen 1.5 ±â€Š0.3 g l at the 2 : 1 : 1 ratio vs. 21 ±â€Š1% and 2.1 ±â€Š0.4 g l respectively at the 1 : 1 : 2 ratio). Compared with FFP at any ratio, maximum clot firmness showed higher values in the groups using fibrinogen concentrate (P < 0.0001), whereas endogenous thrombin potential revealed higher values in the groups using prothrombin complex concentrate (P < 0.0001). CONCLUSION: Use of coagulation factor concentrates for the reconstitution of blood allows for delivery of a higher haematocrit and a higher fibrinogen content compared with FFP. However, prothrombin complex concentrate might result in an unnecessary excess of thrombin generation. Clinical studies are warranted to further investigate these in vitro findings.

Human platelet lysate current standards and future developments.

A state-of-the-art workshop focused on the use of human platelet lysate (HPL) for cell therapy. The meeting established that HPL is used mainly as an adjunct material for ex vivo expansion of mesenchymal stem/progenitor cells (MSCs), where it is successfully used as a substitute for fetal bovine serum. HPL manufacturing as a cell expansion supplement is currently not yet uniformly standardized with regard to platelet source and production methodology. There are very few reports of HPL preparations manufactured specifically for direct clinical use. There exists an urgent need for controlled clinical studies for HPL and for standardization of product definition. Workshop participants also stated a need for consensus minimum release criteria to allow for better product definition and to limit variability in performance. The increasing use of cell-based therapies including MSCs has led to an increasing demand for HPL, either produced in blood establishments or large-scale manufacture by biopharmaceutical companies. The use of pooled donor platelets for HPL production may require the implementation of pathogen inactivation procedures and/or removal steps to improve the safety of advanced cell therapy products. There should also be a requirement for thorough risk assessments and risk mitigation steps, including the qualification of suppliers and identification of ingredients as well as meticulous monitoring of product quality and safety profiles. State-of-the-art regulatory approaches for HPL used for human cell propagation and PRP in direct clinical applications were reviewed.

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells.

Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

Concentrated lyophilized plasma used for reconstitution of whole blood leads to higher coagulation factor activity but unchanged thrombin potential compared with fresh-frozen plasma.

BACKGROUND: During massive hemorrhage, it is recommended to transfuse red blood cells, platelet concentrate, and fresh-frozen plasma in a ratio close to 1:1:1. To avoid the thawing process of fresh frozen plasma, lyophilized plasma (LP) is increasingly used. Evidence is limited on the activity of coagulation factors in reconstituted blood using LP and concentrated LP versions. STUDY DESIGN AND METHODS: Whole blood from ten healthy volunteers was separated into red blood cell, fresh frozen plasma, and platelet concentrate units. Aliquots of red blood cells and plasma concentrate were mixed with either fresh frozen plasma (200 mL) or LP at reconstitution ratios of 2:1:1, 1:1:1, and 1:1:2. LP was used either at the recommended standard volume of 200 mL (LP200) or was more concentrated at volumes of 100 and 50 mL (LP100 and LP50, respectively). The hemostatic capacity of each reconstituted whole blood sample was tested with blood cell counts, standard coagulation tests, factor activity, thrombin generation, and viscoelastic assays. RESULTS: Hematocrit, platelet counts, and fibrinogen levels of the three ratios were similar between FFP200 and LP200 units but were lower compared with the corresponding ratios in LP100 and LP50 units. The activity of procoagulant and anticoagulant factors increased linearly with the increasing plasmatic fraction and, at 1:1:2 ratio, was significantly higher in LP50 units compared with FFP200 and LP200 units. Thrombin generation was similar throughout the four plasma groups at any ratio. CONCLUSIONS: Decreasing the dilution volume of LP facilitates reaching higher hematocrit and coagulation protein levels without a relevant increase in thrombin generation. This is due to preserved balance between procoagulant and anticoagulant factors in the concentrated LP preparations.

Single-mode squeezing in arbitrary spatial modes.

As the generation of squeezed states of light has become a standard technique in laboratories, attention is increasingly directed towards adapting the optical parameters of squeezed beams to the specific requirements of individual applications. It is known that imaging, metrology, and quantum information may benefit from using squeezed light with a tailored transverse spatial mode. However, experiments have so far been limited to generating only a few squeezed spatial modes within a given setup. Here, we present the generation of single-mode squeezing in Laguerre-Gauss and Bessel-Gauss modes, as well as an arbitrary intensity pattern, all from a single setup using a spatial light modulator (SLM). The degree of squeezing obtained is limited mainly by the initial squeezing and diffractive losses introduced by the SLM, while no excess noise from the SLM is detectable at the measured sideband. The experiment illustrates the single-mode concept in quantum optics and demonstrates the viability of current SLMs as flexible tools for the spatial reshaping of squeezed light.

Dying blood mononuclear cell secretome exerts antimicrobial activity.

BACKGROUND: Several activities are attributed to antimicrobial peptides (AMPs), including bacterial killing, leucocyte recruitment and angiogenesis. Despite promises of advanced cellular therapies for treatment of diabetic foot ulcer, it is currently accepted that paracrine factors rather than cellular components are causative for the observed effects. Whether AMPs are present in the mononuclear cell (MNC) secretome (MNC-sec) of white blood cells that are beneficial in experimental wound healing is not known. MATERIALS AND METHODS: Antimicrobial activity of the secretomes of nonirradiated (MNC-sec) and γ-irradiated MNCs (MNC-sec rad) was analysed by microdilution assay. AMPs were determined by quantitative real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Whether human MNC-sec rad causes AMP secretion in vivo was examined in an experimental rat model. Image flow cytometry was used to determine the type of cell death induced in MNCs after exposure to γ-radiation. RESULTS: The antimicrobial activity assay revealed a bactericidal activity of MNC-sec rad and to a lesser degree also of MNC-sec. Image flow cytometry showed that γ-irradiation of MNCs induced early apoptosis followed mainly by necroptosis. RT-PCR and ELISA revealed a high abundance of different AMPs in the secretome of MNCs. In addition, human MNC-sec elicited an increase in de novo endogenous AMP production in rats in vivo. CONCLUSION: We provide evidence that the secretome of MNCs has direct and indirect positive effects on the immune defence system, including augmentation of antibacterial properties. Our data further suggest that necroptosis could play a key role for the release of paracrine factors and the therapeutic action of MNC-sec rad.

Compound heterozygosity of two novel RHAG alleles leads to a considerable disruption of the Rh complex.

BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

The DAU cluster: a comparative analysis of 18 RHD alleles, some forming partial D antigens.

BACKGROUND: The Rh system is the most complex and polymorphic blood group system in humans with more than 460 alleles known for the RHD gene. The DAU cluster of RHD alleles is characterized by the single-nucleotide change producing the p.Thr379Met amino acid substitution. It is called the DAU-0 allele and has been postulated to be the primordial allele, from which all other alleles of the DAU cluster have eventually evolved. STUDY DESIGN AND METHODS: For two novel DAU alleles, the nucleotide sequences of all 10 exons as well as adjacent intronic regions, including the 5' and 3' untranslated regions (UTR), were determined for the RHD and RHCE genes. A phylogenetic tree for all DAU alleles was established using the neighbor-joining method with Pan troglodytes as root. Standard hemagglutination and flow cytometry tests were performed. RESULTS: We characterized two DAU alleles, DAU-11 and DAU-5.1, closely related to DAU-3 and DAU-5, respectively. A phylogenetic analysis of the 18 known DAU alleles indicated point mutations and interallelic recombination contributing to diversification of the DAU cluster. CONCLUSIONS: The DAU alleles encode a group of RhD protein variants, some forming partial D antigens known to permit anti-D in carriers; all are expected to cause anti-D alloimmunization in recipients of red blood cell transfusions. The DAU alleles evolved through genomic point mutations and recombination. These results suggest that the cluster of DAU alleles represent a clade, which is concordant with our previous postulate that they derived from the primordial DAU-0 allele.

Mononuclear cell secretome protects from experimental autoimmune myocarditis.

AIMS: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. METHODS AND RESULTS: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. CONCLUSION: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.

Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

ImmunExplorer (IMEX): a software framework for diversity and clonality analyses of immunoglobulins and T cell receptors on the basis of IMGT/HighV-QUEST preprocessed NGS data.

BACKGROUND: Today's modern research of B and T cell antigen receptors (the immunoglobulins (IG) or antibodies and T cell receptors (TR)) forms the basis for detailed analyses of the human adaptive immune system. For instance, insights in the state of the adaptive immune system provide information that is essentially important in monitoring transplantation processes and the regulation of immune suppressiva. In this context, algorithms and tools are necessary for analyzing the IG and TR diversity on nucleotide as well as on amino acid sequence level, identifying highly proliferated clonotypes, determining the diversity of the cell repertoire found in a sample, comparing different states of the human immune system, and visualizing all relevant information. RESULTS: We here present IMEX, a software framework for the detailed characterization and visualization of the state of human IG and TR repertoires. IMEX offers a broad range of algorithms for statistical analysis of IG and TR data, CDR and V-(D)-J analysis, diversity analysis by calculating the distribution of IG and TR, calculating primer efficiency, and comparing multiple data sets. We use a mathematical model that is able to describe the number of unique clonotypes in a sample taking into account the true number of unique sequences and read errors; we heuristically optimize the parameters of this model. IMEX uses IMGT/HighV-QUEST analysis outputs and includes methods for splitting and merging to enable the submission to this portal and to combine the outputs results, respectively. All calculation results can be visualized and exported. CONCLUSION: IMEX is an user-friendly and flexible framework for performing clonality experiments based on CDR and V-(D)-J rearranged regions, diversity analysis, primer efficiency, and various different visualization experiments. Using IMEX, various immunological reactions and alterations can be investigated in detail. IMEX is freely available for Windows and Unix platforms at http://bioinformatics.fh-hagenberg.at/immunexplorer/.

High dose ionizing radiation regulates micro RNA and gene expression changes in human peripheral blood mononuclear cells.

BACKGROUND: High dose ionizing radiation (IR) induces potent toxic cell effects mediated by either direct DNA damage or the production of reactive oxygen species (ROS). IR-induced modulations in multiple biological processes have been proposed to be partly regulated by radiosensitive microRNA (miRNA). In order to gain new insights into the role of miRNAs in the regulation of biological processes after IR, we have investigated changes in mRNA and miRNA expression after high dose IR. RESULTS: IR induced changes in the mRNA and miRNA profiles of human peripheral blood mononuclear cells (PBMCs). When comparing non-irradiated and irradiated samples, we detected a time-dependent increase in differentially expressed mRNAs and miRNAs, with the highest differences detectable 20 hours after exposure. Gene ontology analysis revealed that very early events (up to 4 hours) after irradiation were specifically associated with p53 signaling and apoptotic pathways, whereas a large number of diverse cellular processes were deregulated after 20 hours. Transcription factor analysis of all up-regulated genes confirmed the importance of p53 in the early post-irradiation phase. When analyzing miRNA expression, we found 177 miRNAs that were significantly regulated in the late post-irradiation phase. Integrating miRNA and target gene expression data, we found a significant negative correlation between miRNA-mRNA and identified hepatic leukemia factor (HLF) as a transcription factor down-regulated in the response to IR. These regulated miRNAs and the HLF target genes were involved in modulating radio-responsive pathways, such as apoptosis, the MAKP signaling pathway, endocytosis, and cytokine-cytokine interactions. CONCLUSION: Using a large dataset of mRNA and miRNA expression profiles, we describe the interplay of mRNAs and miRNAs in the regulation of gene expression in response to IR at a posttranscriptional level and their involvement in the modulation of radiation-induced biological pathways.

Qualifying high-throughput immune repertoire sequencing.

Diversity of B and T cell receptors, achieved by gene recombination and somatic hypermutation, allows the immune system for recognition and targeted reaction against various threats. Next-generation sequencing for assessment of a cell's gene composition and variation makes deep analysis of one individual's immune spectrum feasible. An easy to apply but detailed analysis and visualization strategy is necessary to process all sequences generated. We performed sequencing utilizing the 454 system for CLL and control samples, utilized the IMGT database and applied the presented analysis tools. With the applied protocol, malignant clones are found and characterized, mutational status compared to germline identity is elaborated in detail showing that the CLL mutation status is not as monoclonal as generally thought. On the other hand, this strategy is not solely applicable to the 454 sequencing system but can easily be transferred to any other next-generation sequencing platform.

Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells.

BACKGROUND AIMS: As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells. METHODS: The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs). RESULTS: Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities. CONCLUSIONS: The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs.