Immunogenetics in stem cell donor registry work: The DKMS example (Part 2).

DKMS is a leading stem cell donor registry with more than 9 million donors. Donor registry activities share many touch points with topics from immunogenetics or population genetics. In this two-part review article, we deal with these aspects of donor registry work by using the example of DKMS. In the second part of the review, we focus on donor typing of non-HLA genes, the impact of donor age, gender and CMV serostatus on donation probabilities, the identification of novel HLA, KIR and MIC alleles by high-throughput donor typing, the activities of the Collaborative Biobank and pharmacogenetics in the donor registry context.

Immunogenetics in stem cell donor registry work: The DKMS example (Part 1).

Currently, stem cell donor registries include more than 35 million potential donors worldwide to provide HLA-matched stem cell products for patients in need of an unrelated donor transplant. DKMS is a leading stem cell donor registry with more than 9 million donors from Germany, Poland, the United States, the United Kingdom, India and Chile. DKMS donors have donated hematopoietic stem cells more than 80,000 times. Many aspects of donor registry work are closely related to topics from immunogenetics or population genetics. In this two-part review article, we describe, analyse and discuss these areas of donor registry work by using the example of DKMS. Part 1 of the review gives a general overview on DKMS and includes typical donor registry activities with special focus on the HLA system: high-throughput HLA typing of potential stem cell donors, HLA haplotype frequencies and resulting matching probabilities, and donor file optimization with regard to HLA diversity.

Magnesium corrosion particles do not interfere with the immune function of primary human and murine macrophages.

Magnesium is currently under investigation as a prospective biodegradable implant material. Biodegradation of magnesium causes a release of magnesium, hydroxide ions and hydrogen gas but it can also lead to the formation of particulate debris. Implant-derived particles may have immunotoxic effects. To investigate the influence of magnesium-derived particles on the immune functions of primary macrophages, up to 500 µg/ml magnesium or magnesium corrosion particles were added to the cell culture medium. No major effects were observed on cell viability and on the release of the proinflammatory cytokine tumor necrosis factor (TNF)α. In addition, the ability of macrophages to stimulate proliferation of allogenic lymphocytes in a mixed leukocyte reaction remained unaffected. When macrophages were incubated with magnesium particles and then infected with the apathogenic Mycobacterium smegmatis, infection-induced TNFα secretion from murine macrophages was inhibited but not from human macrophages. However, the bactericidal activity of either cell type was not influenced. In conclusion, magnesium-related particles did not restrict the immune function of macrophages, suggesting that magnesium implants and corrosion particles derived thereof are highly biocompatible and have a low inflammatory potential.

Pyrogen detection methods: comparison of bovine whole blood assay (bWBA) and monocyte activation test (MAT).

BACKGROUND: Pyrogen detection is of utmost importance in pharmaceutical industry, laboratories and health care institutions. As an alternative to the animal-consuming rabbit pyrogen test or Limulus amoebocyte lysate test, the monocyte activation test was introduced as a gold standard method in the European Pharmacopoeia. However, the monocyte activation test has not gained wide acceptance in practice. METHODS: We stimulated bovine whole blood with different endotoxin preparations (lipopolysaccharide E.coli 0127:B8 and 0113:H10), as well as the non-endotoxin pyrogens peptidoglycan and lipoteichoic acid. Prostaglandin E2 (PGE2) served as read out. RESULTS: Employing PGE2 as read out enabled detection limits of 0.04 EU/ml for lipopolysaccharide 0127:B8, 0.25 EU/ml for lipopolysaccharide 0113:H10 and 10 µg/ml of lipoteichoic acid as well as peptidoglycan. To evaluate the bWBA test system as a possible alternative to the MAT we performed a peer-to-peer comparison of the two methods and confirmed similar sensitivities. CONCLUSIONS: In conclusion, the bovine whole blood assay (bWBA) reproducibly enabled sensitive detection of endotoxin and non-endotoxin pyrogens and may thus become a viable alternative for pyrogen testing.

Increased accumulation of magnetic nanoparticles by magnetizable implant materials for the treatment of implant-associated complications.

BACKGROUND: In orthopaedic surgery, accumulation of agents such as anti-infectives in the bone as target tissue is difficult. The use of magnetic nanoparticles (MNPs) as carriers principally enables their accumulation via an externally applied magnetic field. Magnetizable implants are principally able to increase the strength of an externally applied magnetic field to reach also deep-seated parts in the body. Therefore, the integration of bone-addressed therapeutics in MNPs and their accumulation at a magnetic orthopaedic implant could improve the treatment of implant related infections. In this study a martensitic steel platelet as implant placeholder was used to examine its accumulation and retention capacity of MNPs in an in vitro experimental set up considering different experimental frame conditions as magnet quantity and distance to each other, implant thickness and flow velocity. RESULTS: The magnetic field strength increased to approximately 112% when a martensitic stainless steel platelet was located between the magnet poles. Therewith a significantly higher amount of magnetic nanoparticles could be accumulated in the area of the platelet compared to the sole magnetic field. During flushing of the tube system mimicking the in vivo blood flow, the magnetized platelet was able to retain a higher amount of MNPs without an external magnetic field compared to the set up with no mounted platelet during flushing of the system. Generally, a higher flow velocity led to lower amounts of accumulated MNPs. A higher quantity of magnets and a lower distance between magnets led to a higher magnetic field strength. Albeit not significantly the magnetic field strength tended to increase with thicker platelets. CONCLUSION: A martensitic steel platelet significantly improved the attachment of magnetic nanoparticles in an in vitro flow system and therewith indicates the potential of magnetic implant materials in orthopaedic surgery. The use of a remanent magnetic implant material could improve the efficiency of capturing MNPs especially when the external magnetic field is turned off thus facilitating and prolonging the effect. In this way higher drug levels in the target area might be attained resulting in lower inconveniences for the patient.

Regional HLA differences in Poland and their effect on stem cell donor registry planning.

Regional HLA frequency differences are of potential relevance for the optimization of stem cell donor recruitment. We analyzed a very large sample (n = 123,749) of registered Polish stem cell donors. Donor figures by 1-digit postal code regions ranged from n = 5,243 (region 9) to n = 19,661 (region 8). Simulations based on region-specific haplotype frequencies showed that donor recruitment in regions 0, 2, 3 and 4 (mainly located in the south-eastern part of Poland) resulted in an above-average increase of matching probabilities for Polish patients. Regions 1, 7, 8, 9 (mainly located in the northern part of Poland) showed an opposite behavior. However, HLA frequency differences between regions were generally small. A strong indication for regionally focused donor recruitment efforts can, therefore, not be derived from our analyses. Results of haplotype frequency estimations showed sample size effects even for sizes between n≈5,000 and n≈20,000. This observation deserves further attention as most published haplotype frequency estimations are based on much smaller samples.

Synovial distribution of "systemically" administered acetylsalicylic acid in the isolated perfused equine distal limb.

BACKGROUND: This study investigated synovial concentrations of acetylsalicylic acid (ASA) and its metabolite salicylic acid (SA) in the equine fetlock joint following systemic administration of ASA. Salicylates were chosen because SA is the only nonsteroidal anti-inflammatory drug for which threshold levels exist for plasma and urine in equine sports. To avoid animal experiments, the study was conducted using an ex vivo model of the isolated perfused equine distal limb in combination with plasma concentrations obtained from literature.Salicylate concentrations in the joint were determined using microdialysis and high performance liquid chromatography (HPLC). Any anti-inflammatory effect of synovial ASA concentrations was assessed using an ASA EC50 (half maximal effective concentration) determined in equine whole blood. RESULTS: The ASA concentration in the synovial fluid (n=6) reached a maximum of 4 µg/mL, the mean concentration over the entire perfusion period was 2 µg/mL. Maximum SA concentration was 17 µg/mL, the average was 14 µg/mL. ASA and SA concentration in the synovial fluid exceeded systemic concentrations 2 h and 3.5 h after "systemic" administration, respectively. CONCLUSIONS: ASA and SA accumulated in the in the synovial fluid of the ex vivo model despite decreasing systemic concentrations. This suggests a prolonged anti-inflammatory effect within the joint that remains to be further elucidated.

The use of an in vitro-cultured porcine nasal mucosa model for the biocompatibility assessment of biodegradable magnesium.

The development of an in vitro-cultured porcine nasal mucosa model is described. The model was subsequently used for the biocompatibility testing of resorbable magnesium-based implants, which are intended for use in the nasal cavity of patients with chronic rhinosinusitis (CRS). Test specimens made from either pure magnesium or titanium were incubated with the mucosal tissue for 48 hours. Afterwards, tissue viability, PGE2, IL-6 and IL-8 release, magnesium ion release, succinate dehydrogenase activity, apoptosis and 14C amino acid incorporation, were determined. The results suggested favourable biocompatibility, even in the case of rapidly-degrading pure magnesium. However, presumed effects on protein synthesis and apoptosis could not be confirmed.

High-resolution human leukocyte antigen allele and haplotype frequencies of the Polish population based on 20,653 stem cell donors.

We present high-resolution allele and haplotype frequency (HF) estimations of the Polish population based on more than 20,000 registered stem cell donors. Sequencing-based donor human leukocyte antigen (HLA) typing led to unambiguous typing results in most cases (between 94.3% for HLA-DRB1 and 96.9% for HLA-B). HF estimations were carried out with a new, validated implementation of the expectation-maximization algorithm that allowed processing of data with ambiguities. Our results confirm several earlier results, for example, the relative commonness of the haplotype A*25:01 g, B*18:01 g, C*12:03, DRB1*04:01 in the Polish population. Because of the large sample size, we were able to obtain results of unprecedented accuracy. The estimated population-specific HFs were then used to analyze questions of strategic donor registry planning. Simulated matching probabilities by donor file size suggest that there is a need for intense donor recruitment efforts in Poland despite the large German donor registry and the genetic relatedness of both populations. Based on the current German registry size of approximately 4 million donors, the recruitment of 100,000 Polish donors would produce a stronger increase in matching probabilities for Polish patients than the recruitment of 3.3 million additional German donors.

Ex vivo examination of the biocompatibility of biodegradable magnesium via microdialysis in the isolated perfused bovine udder model.

PURPOSE: Being biodegradable, magnesium is considered a promising future implant material but very little is known about the biocompatibility for the tissues in direct contact with it. In this study, the degradation of pure magnesium implants in the skin of an isolated bovine udder was examined over a period of five hours. METHODS: Microdialysis technique was used in order to investigate the reactions at the interface of implant and tissue. Pure titanium implants served as control. Degradation behavior and biocompatibility were evaluated via extracellular magnesium ion concentration and PGE2 and TNF alpha served as indicators of inflammation. RESULTS: Concentrations of 5.5 mmol/l Mg2+ were detected at the beginning, which decreased to a plateau of about 3.5 mmol/l after approximately two and a half hours. PGE2 and TNF alpha concentrations indicated no major inflammatory tissue response to the degradation. CONCLUSIONS: These results give an idea of the ion burden at the implantation site of degrading magnesium and suggest good biocompatibility even at the tissue-implant interface.