Next-generation HLA typing of 382 International Histocompatibility Working Group reference B-lymphoblastoid cell lines: Report from the 17th International HLA and Immunogenetics Workshop

Extended molecular characterization of HLA genes in the IHWG reference B-lymphoblastoid cell lines (B-LCLs) was one of the major goals for the 17th International HLA and Immunogenetics Workshop (IHIW). Although reference B-LCLs have been examined extensively in previous workshops complete high-resolution typing was not completed for all the classical class I and class II HLA genes. To address this, we conducted a single-blind study where select panels of B-LCL genomic DNA samples were distributed to multiple laboratories for HLA genotyping by next-generation sequencing methods. Identical cell panels comprised of 24 and 346 samples were distributed and typed by at least four laboratories in order to derive accurate consensus HLA genotypes. Overall concordance rates calculated at both 2- and 4-field allele-level resolutions ranged from 90.4% to 100%. Concordance for the class I genes ranged from 91.7 to 100%, whereas concordance for class II genes was variable; the lowest observed at HLA-DRB3 (84.2%). At the maximum allele-resolution 78 B-LCLs were defined as homozygous for all 11 loci. We identified 11 novel exon polymorphisms in the entire cell panel. A comparison of the B-LCLs NGS HLA genotypes with the HLA genotypes catalogued in the IPD-IMGT/HLA Database Cell Repository, revealed an overall allele match at 68.4%. Typing discrepancies between the two datasets were mostly due to the lower-resolution historical typing methods resulting in incomplete HLA genotypes for some samples listed in the IPD-IMGT/HLA Database Cell Repository. Our approach of multiple-laboratory NGS HLA typing of the B-LCLs has provided accurate genotyping data. The data generated by the tremendous collaborative efforts of the 17th IHIW participants is useful for updating the current cell and sequence databases and will be a valuable resource for future studies

Is platelet-activating factor produced during hemodialysis with AN-69 polyacrylonitrile membrane?

Background: Platelet-activating factor (PAF) production during hemodialysis (HD) with cuprophane (CU) membrane has previously been demonstrated, while the results regarding PAF production during HD with AN-69 polyacrylonitrile membrane are dubious. In this study an attempt is made to show that PAF is produced during HD using AN-69 membrane while comparing this production with the corresponding one from HD with CU. Since previous studies have indicated that PAF, like the complement system, could also be implicated in HD-related leukopenia and thrombocytopenia (especially when CU membrane is used), the circulating leukocyte and platelet counts as well as the C3a-desArg and SC5b-9 (soluble, nonlytic form of the terminal complement complex) levels were measured. Methods: Ten hemodialyzed patients were subjected to HD with CU and AN-69 membranes for 2 consecutive weeks (first week with CU and second with AN-69). During the third HD session of each week and at different times (0, 2, 5, 15, 30, 60, 180 and 240 min), the PAF levels in the blood as well as the leukocyte and platelet counts were measured, while the circulating levels of the C3a-desArg and SC5b-9 were measured at 0, 5, 15, 60 and 240 min. PAF was detected by ethanol extraction, followed by purification by column chromatography and high-pressure liquid chromatography and finally quantified by bioassay. The C3a-desArg and SC5b-9 fractions of the complement were measured by immunoassay while an autoanalyzer gave the leukocyte and platelet counts. Results: Circulating PAF levels were detected at all time intervals during HD with AN-69 (PAFAN-69) and CU (PAFCU) membranes. At all time intervals PAFAN-69 <PAFCU, however, statistically significant differences (s) between the two membranes existed only at 15, 30, 60, 180 and 240 min. The highest PAFAN-69 and PAFCU occurred at 5 and 15 min into dialysis, respectively. The same observations were made for circulating C3a-desArg levels (s existed additionally at 5 min as well). The reduction of the circulating leukocytes had almost a mirror image with the C3a-desArg as well as PAF levels while the maximal reduction of platelets was observed after 2 min into dialysis with both membranes (i.e., simultaneously with the first increase in PAF secretion). Conclusions: PAF is indeed produced during HD with AN-69 membrane, as it is during HD with CU. At all time intervals during the HD procedure, PAFAN-69 < PAFCU. PAF seems to contribute to HD-related leukopenia and thrombocytopenia with both membranes. © 2002 S. Karger AG, Basel

Decellularized human umbilical artery used as nerve conduit

Treatment of injuries to peripheral nerves after a segmental defect is one of the most challenging surgical problems. Despite advancements in microsurgical techniques, complete recovery of nerve function after repair has not been achieved. The purpose of this study was to evaluate the use of the decellularized human umbilical artery (hUA) as nerve guidance conduit. A segmental peripheral nerve injury was created in 24 Sprague-Dawley rats. The animals were organized into two experimental groups with different forms of repair: decellularized hUA (n = 12), and autologous nerve graft (n = 12). Sciatic faction index and gastrocnemius muscle values were calculated for functional recovery evaluation. Nerve morphometry was used to analyze nerve regeneration. Results showed that decellularized hUAs after implantation were rich in nerve fibers and characterized by improved Sciatic Functional index (SFI) values. Decellularized hUA may support elongation and bridging of the 10 mm nerve gap. © 2018 by the authors

Effect of Cord Blood Platelet Gel on wound healing capacity of human Mesenchymal Stromal Cells

Background: Wound healing is a dynamic process, involving the recruitment of growth factors, cytokines, chemokines and cellular populations. Recently, the Cord Blood Platelet Gel (CBPG) has been applied successfully in wound closure and tissue regeneration. Moreover, its proper combination with stem cell populations such as Mesenchymal Stromal Cells (MSCs) may positively improve the wound healing process. Based on the above data, this study aimed to the evaluation of wound healing capacity of MSCs combined with CBPG under in vitro conditions. Methods: Initially, CBPG was developed from Cord Blood Units (CBUs). The determination of wound healing ability of MSCs was performed using the scratch wound assay. In addition, the morphological features, immunophenotypical characteristics and differentiation capacity of MSCs were evaluated. Results: Scratch wound assay results showed, that CBPG could positively stimulate the MSCs migration. Moreover, MSCs cultured in presence of CBPG were characterized by elongated shape and improved stemness properties as it was indicated by flow cytometric analysis and differentiation process. Conclusion: These results clearly showed the beneficial effect of CBPG in combination with MSCs in wound healing. The proper combination of CBPG with stem cells strategy may enhance the healing process in patients with skin erosions. © 2020 Elsevier Lt

The incidence of 21 alpha-hydroxylase deficiency in Greek hyperandrogenic women: screening and diagnosis

The purpose of this prospective study was to determine the incidence of any form of 21 alpha-hydroxylase deficiency among Greek women with hyperandrogenic symptoms, and to test the predictive value of basal serum 17-hydroxyprogesterone (17-OHP) in the early follicular phase as a screening index for patient preselection to adrenocorticotropic hormone (ACTH) testing. Eighty-eight unselected women with hyperandrogenic symptoms were examined in the Gynecological Endocrinology Unit of the Second Department of Obstetrics and Gynecology of Athens University. Using the ACTH-stimulated 17-OHP values at 60 minutes (17-OHP60) the study population was divided into four groups (A, B, C and D). Clinical and basal hormonal parameters as well as serum 17-OHP60 values and human leukocyte antigens were studied. Both clinical and basal hormonal parameters could be used to distinguish only patients with severe 21 alpha-hydroxylase deficiency (group A). In contrast, patients with moderate non-classical congenital adrenal hyperplasia (NC-CAH; group B), heterozygotes for NC-CAH (group C), and unaffected females (group D) can be diagnosed and classified only by serum 17-OHP60 values. In conclusion, the incidence of NC-CAH in creek females with hyperandrogenic symptoms is 5.4%. The positive predictive value of basal 17-OHP is only 13% for this disease. Only 17-OHP60 helps to diagnose and classify moderate and mild forms of NC-CAH. Thus, it seems that ACTH testing is imperative in every subject suspected of this enzymatic disorder

Evaluation of a decellularization protocol for the development of a decellularized tracheal scaffold

Background/Aim: Currently, there are no effective solutions for the treatment of advanced disorders of the airways. The aim of this study was to assess the efficacy of a decellularization protocol of the trachea in order to produce a functional scaffold for serious clinical respiratory disorders. Materials and Methods: Rat tracheas were decellularized using a protocol which included constituents with chemical action. Histological analysis was performed in order to evaluate the efficacy of decellularization. Genetic material was assayed and the toxicity of the decellularization protocol was assessed. Results: Histological analysis confirmed the removal of the nuclear and cellular components of the decellularized tissue, as well as maintenance of the extracellular matrix. DNA quantification showed removal of the genetic material. Furthermore, the decellularization protocol did not induce any cytotoxicity on tracheaI tissue. Conclusion: The decellularization protocol was effective for tracheal decellularization. The final aim, in the future, would be to create a tissue-engineered airway which will be able to function normally. © 2019 International Institute of Anticancer Research. All Rights Reserved

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering

The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations. Mesenchymal stromal cells (MSCs) derived from the Wharton's Jelly (WJ) tissue can be used as a source for obtaining vascular smooth muscle cells (VSMCs), while the human umbilical arteries (hUAs) can serve as a scaffold for blood vessel engineering. © 2020 Baishideng Publishing Group Inc

Optimizing isolation culture and freezing methods to preserve Wharton's jelly's mesenchymal stem cell (MSC) properties: An MSC banking protocol validation for the Hellenic Cord Blood Bank

Background Mesenchymal stem or stromal cells (MSCs) are a heterogeneous population that can be isolated from many tissues including umbilical cord Wharton's jelly (UC-WJ). Although initially limited in studies such as a hematopoietic stem cell transplantation adjuvant, an increasing number of clinical trials consider MSCs as a potential anti-inflammatory or a regenerative medicine agent. It has been proposed that creating a repository of MSCs would increase their availability for clinical applications. The aim of this study was to assess the optimal isolation and cryopreservation procedures to facilitate WJ MSC banking. Study Design and Methods Cells were isolated from UC-WJ using enzymatic digestion or plastic adhesion methods. Their isolation efficacy, growth kinetics, immunophenotype, and differentiation potential were studied, as well as the effects of freezing. Flow cytometry for common MSC markers was performed on all cases and differentiation was shown with histocytochemical staining. Finally, the isolation efficacy on cryopreserved WJ tissue fragments was tested. Conclusion These data showed that viable MSCs can only be isolated from fresh UC-WJ tissue, setting the foundation for clinical-grade banking. Results MSC isolation was successful using both isolation methods on fresh UC-WJ tissue. However, UC-WJ MSC isolation from frozen tissue fragments was impossible. Flow cytometry analysis revealed that only MSC markers were expressed on the surface of the isolated cells while differentiation assays showed that they were capable of trilinear differentiation. All the above characteristics were also preserved in isolated UC-WJ MSCs over the cryopreservation study period. © 2014 AABB