Characterization of three novel HLA class I alleles, HLA-A*31:01:02:56, HLA-B*35:588 and HLA-B*57:153.

Characterization of three novel HLA class I alleles by next generation sequencing.

Functional Assessment of Vitamin D Status by a Novel Metabolic Approach: The Low Vitamin D Profile Concept.

BACKGROUND: Determining serum 25-hydroxyvitamin D [25(OH)D], 24,25-dihydroxyvitamin D [24,25(OH)2D] and the vitamin D metabolite ratio (VMR) allows the identification of individuals with a low vitamin D metabolite profile. Here, we evaluated if such a functional approach provides superior diagnostic information to serum 25(OH)D alone. METHODS: 25(OH)D, 24,25(OH)2D, and the VMR were determined in participants of the DESIRE (Desirable Vitamin D Concentrations, n = 2010) and the LURIC (Ludwigshafen Risk and Cardiovascular Health, n = 2456) studies. A low vitamin D metabolite profile (vitamin D insufficiency) was defined by a 24,25(OH)2D concentration <1.2 ng/mL (<3 nmol/L) and a VMR <4%. Parathyroid hormone (PTH) and bone turnover markers were measured in both cohorts, whereas 10-year mortality data was recorded in LURIC only. RESULTS: The median age in DESIRE and LURIC was 43.3 and 63.8 years, respectively. Median 25(OH)D concentrations were 27.2 ng/mL (68.0 nmol/L) and 15.5 ng/mL (38.8 nmol/L), respectively. Serum 25(OH)D deficiency, defined as <20.2 ng/mL (<50 nmol/L), was present in 483 (24.0%) and 1701 (69.3%) participants of DESIRE and LURIC, respectively. In contrast, only 77 (3.8%) and 521 (21.2%) participants had a low vitamin D metabolite profile. Regardless of the serum 25(OH)D concentration, a low vitamin D metabolite profile was associated with a significantly higher PTH, accelerated bone metabolism, and higher all-cause mortality than an unremarkable vitamin D metabolite profile. CONCLUSIONS: The personalized assessment of vitamin D status using a functional approach better identifies patients with accelerated bone metabolism and increased mortality than the use of a fixed 25(OH)D cutoff of 20 ng/mL (50 nmol/L).

Pilot Study to Gain First Indications for the Impact of a 3-Month's Oral Intake of a Sucrosomial Iron Supplement on Hemoglobin in Iron-Deficient Blood Donors.

Introduction: Regular whole blood donors often suffer from iron deficiency (ID) or iron deficiency anemia due to the loss of 200-300 mg of iron with each donation. Hemoglobin (Hb) as donor eligibility criterion reflects iron stores only poorly. ID in blood donors is typically prevented or treated with orally administered ferrous salts, which frequently cause gastrointestinal side effects. A high daily oral iron dose is counterproductive due to hepcidin upregulation. Oral sucrosomial iron (sucriron) is encapsulated ferric pyrophosphate that may be an option for blood donors due to its supposed high bioavailability and good tolerability. Methods: This monocentric single-cohort pilot study included fifty whole blood donors (divided into premenopausal women, postmenopausal women, and men) who did not meet Hb donation criteria. Participants aged 18-65 years with ferritin <30 ng/mL and venous Hb <12.5 g/dL in women and Hb <13.5 g/dL in men received oral sucriron (30 mg iron) for 90-120 days. Primary endpoints were the increase of Hb and ferritin. Results: Forty-seven participants completed the study. With the limitation that no control group was included, there was a substantial overall median increase of 0.94 g/dL Hb and 4.97 ng/mL ferritin (standardized on 90 days of iron intake). These value improvements were likewise observed in each of the subgroups. sucriron was very well tolerated, with almost no gastrointestinal side effects identified. Conclusion: A clear increase of Hb and ferritin was observed after the intake of sucriron, so it may be a reasonable and useful alternative to traditional oral iron therapy. The ease of administration, pleasant taste, dietary supplement status, and, most importantly, good tolerability highlight the value of sucriron supplementation.

Platelet-Rich Fibrin in Oral Surgery and Implantology: A Narrative Review.

Background: The application of blood concentrates has gained popularity in dentistry in recent years. Platelet-rich fibrin (PRF) has been discussed frequently due to a high content of growth factors and the option of chair-side manufacturing in a simple centrifugation process. PRF is free from adjuvants and inexpensive to produce. The number of studies reporting beneficial effects of PRF in various clinical applications such as alveolar ridge preservation, sinus floor elevation, management and prevention of medical-related osteonecrosis of the jaw, third molar extractions, and guided bone regeneration in dentistry has increased recently. However, to date, neither clinical recommendations nor guidelines are available. The present narrative review aims to summarize the level of evidence on the clinical application of PRF within the field of oral surgery and implantology. Summary: A literature search in Pubmed and Medline has identified 34 articles as a basis for this narrative review. The effectiveness of the clinical application of PRF has been analyzed for five indications within dentistry: medical-related osteonecrosis of the jaw, wisdom tooth extraction, guided bone regeneration, sinus floor elevation, and alveolar ridge preservation. The amount of data for third molar extractions, socket preservation, and guided bone regeneration is extensive. Less data were available for the use of PRF in combination with sinus floor elevations. There is a lack of studies with scientific evidence on PRF and medical-related osteonecrosis of the jaw; however, studies positively impact patient-related outcome measures. Most studies report on beneficial effects when PRF is additionally applied in intrabony defects. There is no evidence of the positive effects of PRF combined with bone graft materials during sinus floor elevation. However, some benefits are reported with PRF as a sole filling material. Key Messages: Many recently published studies show the positive clinical impact of PRF. Yet, further research is needed to ensure the validity of the evidence.

Altered cellular immune response to vaccination against SARS-CoV-2 in patients suffering from autoimmunity with B-cell depleting therapy.

B-cell depleting therapies result in diminished humoral immunity following vaccination against COVID-19, but our understanding on the impact on cellular immune responses is limited. Here, we performed a detailed analysis of cellular immunity following mRNA vaccination in patients receiving B-cell depleting therapy using ELISpot assay and flow cytometry. Anti-SARS-CoV-2 spike receptor-binding domain antibody assays were performed to elucidate B-cell responses. To complement our cellular analysis, we performed immunophenotyping for T- and B-cell subsets. We show that SARS-CoV-2 vaccination using mRNA vaccines elicits cellular T-cell responses in patients under B-cell depleting therapy. Some facets of this immune response including TNFα production of CD4+ T-cells and granzyme B production of CD8+ T-cells, however, are distinctly diminished in these patients. Consequently, it appears that the finely coordinated process of T-cell activation with a uniform involvement of CD4+ and CD8+ T-cells as seen in HCs is disturbed in autoimmune patients. In addition, we observed that immune cell composition does impact cellular immunity as well as sustainability of anti-spike antibody titers. Our data suggest disturbed cellular immunity following mRNA vaccination in patients treated with B-cell depleting therapy. Immune cell composition may be an important determinant for vaccination efficacy.

Biomechanical properties of native and cultured red blood cells-Interplay of shape, structure and biomechanics.

Modern medicine increases the demand for safe blood products. Ex vivo cultured red blood cells (cRBC) are eagerly awaited as a standardized, safe source of RBC. Established culture models still lack the terminal cytoskeletal remodeling from reticulocyte to erythrocyte with changes in the biomechanical properties and interacts with membrane stiffness, viscosity of the cytoplasm and the cytoskeletal network. Comprehensive data on the biomechanical properties of cRBC are needed to take the last step towards translation into clinical use in transfusion medicine. Aim of the study was the comparative analysis of topographical and biomechanical properties of cRBC, generated from human CD34+ adult hematopoietic stem/progenitor cells, with native reticulocytes (nRET) and erythrocytes (nRBC) using cell biological and biomechanical technologies. To gain the desired all-encompassing information, a single method was unsatisfactory and only the combination of different methods could lead to the goal. Topographical information was matched with biomechanical data from optical tweezers (OT), atomic force microscopy (AFM) and digital holographic microscopy (DHM). Underlying structures were investigated in detail. Imaging, deformability and recovery time showed a high similarity between cRBC and nRBC. Young's modulus and plasticity index also confirmed this similarity. No significant differences in membrane and cytoskeletal proteins were found, while lipid deficiency resulted in spherical, vesiculated cells with impaired biomechanical functionality. The combination of techniques has proven successful and experiments underscore a close relationship between lipid content, shape and biomechanical functionality of RBC.

Growth Factor Release within Liquid and Solid PRF.

Aim: The purpose of this study was to obtain data concerning growth factor release within liquid and solid platelet-rich fibrin (PRF) matrices and to estimate the amount of potential interindividual variations as a basis for further preclinical and clinical trials. Therefore, we aimed to determine possible differences in the release of growth factors between liquid and solid PRF. Materials and Methods: Blood samples obtained from four subjects were processed to both liquid and solid PRF matrices using a standard centrifugation protocol. Five growth factors (vascular endothelial growth factor, VEGF; epidermal growth factor, EGF; platelet-derived growth factor-BB, PDGF-BB; transforming growth factor-ß1, TGF-ß1; and matrix metallopeptidase 9, MMP-9) have been evaluated at six time points by ELISA over a total observation period of 10 days (1 h, 7 h, 1 d, 2 d, 7 d, and 10 d). Results: Growth factor release could be measured in all samples at each time point. Comparing liquid and solid PRF matrices, no significant differences were detected (p > 0.05). The mean release of VEGF, TGFß-1, PDGF-BB, and MMP-9 raised to a peak at time point five (day 7) in both liquid and solid PRF matrices. VEGF release was lower in liquid PRF than in solid PRF, whereas those of PDGF-BB and MMP-9 were higher in liquid PRF than in solid PRF at all time points. EGF had its peak release already at time point two after 7 h in liquid and solid matrices (hour 7 EGF solid: mean = 180 pg/mL, SD = 81; EGF liquid: mean = 218 pg/mL, SD = 64), declined rapidly until day 2, and had a second slight peak on day 7 in both groups (day 7 EGF solid: mean = 182 pg/mL, SD = 189; EGF liquid: mean = 81 pg/mL, SD = 70). Conclusions: This study detected growth factor release within liquid and solid PRF matrices with little variations. Further preclinical trials are needed to precisely analyze the growth factor release in larger samples and to better understand their effects on wound healing in different clinical indications.

Generation of the human erythroblast-derived iPSC line UBTi001-A.

We performed reprogramming of human erythroblasts derived from CD34+ hematopoietic stem / progenitor cells of a healthy donor. CD34+ cells were differentiated in-vitro into a pure population of CD36+ erythroblasts and nucleofected with four episomal plasmids expressing SOX2, OCT3/4, KLF4, LIN28, L-MYC and TP53-shRNA. The established iPSC line showed normal karyotype. Pluripotency was confirmed by expression of pluripotency markers and in-vitro differentiation into tissues of all three germ layers. The UBTi001-A iPSC line might provide an attractive source for developmental research on human hematopoiesis and erythropoiesis.

Membrane Properties of Human Induced Pluripotent Stem Cell-Derived Cultured Red Blood Cells.

Cultured red blood cells from human induced pluripotent stem cells (cRBC_iPSCs) are a promising source for future concepts in transfusion medicine. Before cRBC_iPSCs will have entrance into clinical or laboratory use, their functional properties and safety have to be carefully validated. Due to the limitations of established culture systems, such studies are still missing. Improved erythropoiesis in a recently established culture system, closer simulating the physiological niche, enabled us to conduct functional characterization of enucleated cRBC_iPSCs with a focus on membrane properties. Morphology and maturation stage of cRBC_iPSCs were closer to native reticulocytes (nRETs) than to native red blood cells (nRBCs). Whereas osmotic resistance of cRBC_iPSCs was similar to nRETs, their deformability was slightly impaired. Since no obvious alterations in membrane morphology, lipid composition, and major membrane associated protein patterns were observed, reduced deformability might be caused by a more primitive nature of cRBC_iPSCs comparable to human embryonic- or fetal liver erythropoiesis. Blood group phenotyping of cRBC_iPSCs further confirmed the potency of cRBC_iPSCs as a prospective device in pre-transfusional routine diagnostics. Therefore, RBC membrane analyses obtained in this study underscore the overall prospects of cRBC_iPSCs for their future application in the field of transfusion medicine.

Lower Levels of ABO Anti-A and Anti-B of IgM, IgG and IgA Isotypes in the Serum but Not the Saliva of COVID-19 Convalescents.

Individuals with ABO type O, naturally possessing anti-A and anti-B antibodies in their serum, are underrepresented among patients infected with SARS-CoV-2 compared with healthy controls. The ABO antibodies might play a role in the viral transmission. Therefore, we aimed to quantify anti-A/anti-B, including their subclasses IgM, IgG and IgA, in the serum and saliva of Caucasians (n = 187) after mild COVID-19 to compare them with individuals who had never been infected with SARS-CoV-2. Two samples were collected within two months after the diagnosis (median days: 44) and two months later. ABO antibodies were determined by flow cytometry. Additionally, total IgA in saliva and antibodies specific to SARS-CoV-2 were tested by ELISA. COVID-19 convalescents had significantly lower levels of anti-A/anti-B IgM, IgG and IgA in their serum than control subjects (p < 0.001). Interestingly, no significant differences were observed in saliva. ABO antibody levels remained stable over the period considered. No relation of ABO to the level of SARS-CoV-2-specific antibodies was observed. Total IgA was lower in convalescents than in controls (p = 0.038). Whereas ABO antibodies in the saliva may not contribute to the pathogenesis of COVID-19, individual pre-existing high serum concentrations of anti-A/anti-B may have a protective effect against SARS-CoV-2 infection.

Preparation and Storage of Cryoprecipitate Derived from Amotosalen and UVA-Treated Apheresis Plasma and Assessment of In Vitro Quality Parameters.

Cryoprecipitate is a plasma-derived blood product, enriched for fibrinogen, factor VIII, factor XIII, and von Willebrand factor. Due to infectious risk, the use of cryoprecipitate in Central Europe diminished over the last decades. However, after the introduction of various pathogen-reduction technologies for plasma, cryoprecipitate production in blood centers is a feasible alternative to pharmaceutical fibrinogen concentrate with a high safety profile. In our study, we evaluated the feasibility of the production of twenty-four cryoprecipitate units from pools of two units of apheresis plasma pathogen reduced using amotosalen and ultraviolet light A (UVA) (INTERCEPT® Blood System). The aim was to assess the compliance of the pathogen-reduced cryoprecipitate with the European Directorate for the Quality of Medicines (EDQM) guidelines and the stability of coagulation factors after frozen (≤-25 °C) storage and five-day liquid storage at ambient temperature post-thawing. All pathogen-reduced cryoprecipitate units fulfilled the European requirements for fibrinogen, factor VIII and von Willebrand factor content post-preparation. After five days of liquid storage, content of these factors exceeded the minimum values in the European requirements and the content of other factors was sufficient. Our method of production of cryoprecipitate using pathogen-reduced apheresis plasma in a jumbo bag is feasible and efficient.

Identification of the novel HLA-B*51:01:01:78 allele and confirmation of extended sequence of B*18:01:26.

HLA-B*51:01:01:78 and the extended confirmatory sequence of HLA-B*18:01:26 were identified by next generation sequencing.

Humoral immune response to COVID-19 vaccination in diabetes is age-dependent but independent of type of diabetes and glycaemic control: The prospective COVAC-DM cohort study.

AIMS: To investigate the seroconversion following first and second COVID-19 vaccination in people with type 1 and type 2 diabetes in relation to glycaemic control prior to vaccination and to analyse the response in comparison to individuals without diabetes. MATERIALS AND METHODS: This prospective, multicentre cohort study analysed people with type 1 and type 2 diabetes and a glycated haemoglobin level ≤58 mmol/mol (7.5%) or >58 mmol/mol (7.5%), respectively, and healthy controls. Roche's Elecsys anti-SARS-CoV-2 S immunoassay targeting the receptor-binding domain was used to quantify anti-spike protein antibodies 7 to 14 days after the first and 14 to 21 days after the second vaccination. RESULTS: A total of 86 healthy controls were enrolled in the study, as well as 161 participants with diabetes, of whom 150 (75 with type 1 diabetes and 75 with type 2 diabetes) were eligible for the analysis. After the first vaccination, only 52.7% of participants in the type 1 diabetes group and 48.0% of those in the type 2 diabetes group showed antibody levels above the cut-off for positivity. Antibody levels after the second vaccination were similar in participants with type 1 diabetes, participants with type 2 diabetes and healthy controls after adjusting for age, sex and multiple testing (P > 0.05). Age (r = -0.45, P < 0.001) and glomerular filtration rate (r = 0.28, P = 0.001) were significantly associated with antibody response. CONCLUSIONS: Anti-SARS-CoV-2 S receptor-binding domain antibody levels after the second vaccination were comparable in healthy controls and in participants with type 1 and type 2 diabetes, irrespective of glycaemic control. Age and renal function correlated significantly with the extent of antibody levels.

Longitudinal Comparison of Automated SARS-CoV-2 Serology Assays in Assessing Virus Neutralization Capacity in COVID-19 Convalescent Sera.

CONTEXT.­: Serologic tests on automated immunology analyzers are increasingly used to monitor acquired immunity against SARS-CoV-2. The heterogeneity of assays raises concerns about their diagnostic performance and comparability. OBJECTIVE.­: To test sera from formerly infected individuals for SARS-CoV-2 antibodies by using 6 automated serology assays and a pseudoneutralization test (PNT). DESIGN.­: Six SARS-CoV-2 serology assays were used to assess 954 samples collected during a 12-month period from 315 COVID-19 convalescents. The tests determined either antibodies against the viral nucleocapsid (anti-NC) or spike protein (anti-S). Two assays did not distinguish between antibody classes, whereas the others selectively measured immunoglobulin G (IgG) antibodies. PNT was used to detect the presence of neutralizing antibodies. RESULTS.­: Comparison of qualitative results showed only slight to moderate concordance between the assays (Cohen κ < 0.57). Significant correlations (P < .001) were observed between the antibody titers from all quantitative assays. However, titer changes were not detected equally. A total anti-S assay measured an increase in 128 of 172 cases (74%) of a suitable subset, whereas all IgG anti-S tests reported decreases in at least 118 (69%). Regarding the PNT results, diagnostic sensitivities of 89% or greater were achieved with positive predictive values of at least 93%. In contrast, specificity changed substantially over time, varying from 20% to 100%. CONCLUSIONS.­: Comparability of serologic SARS-CoV-2 antibody tests is rather poor. Owing to different diagnostic specificities, the tested assays were not equally capable of capturing changes in antibody titers. However, with thoroughly validated cutoffs, IgG-selective anti-S assays are a reliable surrogate test for SARS-CoV-2 neutralizing antibodies in former COVID-19 patients.

Identification of the novel HLA-C*03:03:01:52N allele, a splice-site variant at the boundary of intron1 and exon2.

HLA-C*03:03:01:52N differs from HLA-C*03:03:01:01 by one nucleotide substitution at position gDNA 202 (A>G) in Intron1.

CD19+IgD+CD27- Naïve B Cells as Predictors of Humoral Response to COVID 19 mRNA Vaccination in Immunocompromised Patients.

Immunocompromised patients are considered high-risk and prioritized for vaccination against COVID-19. We aimed to analyze B-cell subsets in these patients to identify potential predictors of humoral vaccination response. Patients (n=120) suffering from hematologic malignancies or other causes of immunodeficiency and healthy controls (n=79) received a full vaccination series with an mRNA vaccine. B-cell subsets were analyzed prior to vaccination. Two independent anti-SARS-CoV-2 immunoassays targeting the receptor-binding domain (RBD) or trimeric S protein (TSP) were performed three to four weeks after the second vaccination. Seroconversion occurred in 100% of healthy controls, in contrast to 67% (RBD) and 82% (TSP) of immunocompromised patients, while only 32% (RBD) and 22% (TSP) achieved antibody levels comparable to those of healthy controls. The number of circulating CD19+IgD+CD27- naïve B cells was strongly associated with antibody levels (ρ=0.761, P<0.001) and the only independent predictor for achieving antibody levels comparable to healthy controls (OR 1.07 per 10-µL increase, 95%CI 1.02-1.12, P=0.009). Receiver operating characteristic analysis identified a cut-off at ≥61 naïve B cells per µl to discriminate between patients with and without an optimal antibody response. Consequently, measuring of naïve B cells in immunocompromised hematologic patients could be useful in predicting their humoral vaccination response.

MUG Mel3 Cell Lines Reflect Heterogeneity in Melanoma and Represent a Robust Model for Melanoma in Pregnancy.

Melanomas are aggressive tumors with a high metastatic potential and an increasing incidence rate. They are known for their heterogeneity and propensity to easily develop therapy-resistance. Nowadays they are one of the most common cancers diagnosed during pregnancy. Due to the difficulty in balancing maternal needs and foetal safety, melanoma is challenging to treat. The aim of this study was to provide a potential model system for the study of melanoma in pregnancy and to illustrate melanoma heterogeneity. For this purpose, a pigmented and a non-pigmented section of a lymph node metastasis from a pregnant patient were cultured under different conditions and characterized in detail. All four culture conditions exhibited different phenotypic, genotypic as well as tumorigenic properties, and resulted in four newly established melanoma cell lines. To address treatment issues, especially in pregnant patients, the effect of synthetic human lactoferricin-derived peptides was tested successfully. These new BRAF-mutated MUG Mel3 cell lines represent a valuable model in melanoma heterogeneity and melanoma pregnancy research. Furthermore, treatment with anti-tumor peptides offers an alternative to conventionally used therapeutic options-especially during pregnancy.