Superior physical and mental health of healthy volunteers before and five years after mobilized stem cell donation.

BACKGROUND: Safety, tolerability and efficacy of granulocyte colony-stimulating factor (G-CSF) for mobilization of hematopoietic stem and progenitor cells (HSPCs) from healthy donors have been conclusively demonstrated. This explicitly includes, albeit for smaller cohorts and shorter observation periods, biosimilar G-CSFs. HSPC donation is non-remunerated, its sole reward being "warm glow", hence harm to donors must be avoided with maximal certitude. To ascertain, therefore, long-term physical and mental health effects of HSPC donation, a cohort of G-CSF mobilized donors was followed longitudinally. METHODS: We enrolled 245 healthy volunteers in this bi-centric long-term surveillance study. 244 healthy volunteers began mobilization with twice-daily Sandoz biosimilar filgrastim and 242 underwent apheresis after G-CSF mobilization. Physical and mental health were followed up over a period of 5-years using the validated SF-12 health questionnaire. RESULTS: Baseline physical and mental health of HSPC donors was markedly better than in a healthy reference population matched for ethnicity, sex and age. Physical, but not mental health was sharply diminished at the time of apheresis, likely due to side effects of biosimilar G-CSF, however had returned to pre-apheresis values by the next follow-up appointment after 6 months. Physical and mental health slightly deteriorated over time with kinetics reflecting the known effects of aging. Hence, superior physical and mental health compared to the general healthy non-donor population was maintained over time. CONCLUSIONS: HSPC donors are of better overall physical and mental health than the average healthy non-donor. Superior well-being is maintained over time, supporting the favorable risk-benefit assessment of volunteer HSPC donation. Trial registration National Clinical Trial NCT01766934.

Highly efficient CRISPR-Cas9-mediated editing identifies novel mechanosensitive microRNA-140 targets in primary human articular chondrocytes.

OBJECTIVE: MicroRNA 140 (miR-140) is a chondrocyte-specific endogenous gene regulator implicated in osteoarthritis (OA). As mechanical injury is a primary aetiological factor in OA, we investigated miR-140-dependent mechanosensitive gene regulation using a novel CRISPR-Cas9 methodology in primary human chondrocytes. METHOD: Primary (passage 1/2) human OA chondrocytes were isolated from arthroplasty samples (six donors) and transfected with ribonuclear protein complexes or plasmids using single guide RNAs (sgRNAs) targeting miR-140, in combination with Cas9 endonuclease. Combinations of sgRNAs and single/double transfections were tested. Gene editing was measured by T7 endonuclease 1 (T7E1) assay. miRNA levels were confirmed by qPCR in chondrocytes and in wild type murine femoral head cartilage after acute injury. Predicted close match off-targets were examined. Mechanosensitive miR-140 target validation was assessed in 42 injury-associated genes using TaqMan Microfluidic cards in targeted and donor-matched control chondrocytes. Identified targets were examined in RNAseq data from costal chondrocytes from miR-140-/- mice. RESULTS: High efficiency gene editing of miR-140 (90-98%) was obtained when two sgRNAs were combined with double RNP-mediated CRISPR-Cas9 transfection. miR-140 levels fell rapidly after femoral cartilage injury. Of the top eight miR-140 gene targets identified (P < 0.01), we validated three previously identified ones (septin 2, bone morphogenetic protein 2 and fibroblast growth factor 2). Novel targets included Agrin, a newly recognised pro-regenerative cartilage agent, and proteins associated with retinoic acid signalling and the primary cilium. CONCLUSION: We describe a highly efficient CRISPR-Cas9-mediated strategy for gene editing in primary human chondrocytes and identify several novel mechanosensitive miR-140 targets of disease relevance.

CRISPR-Cas9 targeting of MMP13 in human chondrocytes leads to significantly reduced levels of the metalloproteinase and enhanced type II collagen accumulation.

OBJECTIVE: To investigate the efficacy of CRISPR-Cas9 mediated editing in human chondrocytes, and to develop a genome editing approach relevant to cell-based repair. METHODS: Transfection of human articular chondrocytes (both healthy and osteoarthritic) with ribonucleoprotein complexes (RNP) containing Cas9 and a crisprRNA targeting exon2 of MMP13 was performed to assess editing efficiency and effects on MMP13 protein levels and enzymatic activity. Using spheroid cultures, protein levels of a major target of MMP13, type II collagen, were assessed by western blot and immunofluorescence. RESULTS: With an editing efficiency of 63-74%, secreted MMP13 protein levels and activity were significantly reduced (percentage decrease 34.14% without and 67.97% with IL-1ß based on median values of MMP13 enzymatic activity, N = 7) comparing non-edited with edited cell populations using an exon-targeting gRNA resulting in premature stop codons through non-homologous end joining (NHEJ). Accumulation of cartilage matrix protein type II collagen was enhanced in edited cells in spheroid culture, compared to non-edited controls. CONCLUSION: CRISPR-Cas9 mediated genome editing can be used to efficiently and reproducibly establish populations of human chondrocytes with stably reduced expression of key genes of interest without the need for clonal selection. Such an editing approach has the potential to greatly enhance current cell-based therapies for cartilage repair.

Anti-HLA alloantibodies of the IgA isotype in re-transplant candidates part II: Correlation with graft survival.

We reported previously on the widespread occurrence of anti-HLA alloantibodies of the IgA isotype (anti-HLA IgA) in the sera of solid-organ re-transplantation (re-tx) candidates (Arnold et al., ). Specifically focussing on kidney re-tx patients, we now extended our earlier findings by examining the impact of the presence and donor specificity of anti-HLA IgA on graft survival. We observed frequent concurrence of anti-HLA IgA and anti-HLA IgG in 27% of our multicenter collective of 694 kidney re-tx patients. This subgroup displayed significantly reduced graft survival as evidenced by the median time to first dialysis after transplantation (TTD 77 months) compared to patients carrying either anti-HLA IgG or IgA (TTD 102 and 94 months, respectively). In addition, donor specificity of anti-HLA IgA had a significant negative impact on graft survival (TTD 74 months) in our study. Taken together, our data strongly indicate that presence of anti-HLA IgA, in particular in conjunction with anti-HLA-IgG, in sera of kidney re-tx patients is associated with negative transplantation outcome.

General anaesthesia-induced anaphylaxis: impact of allergy testing on subsequent anaesthesia.

BACKGROUND: Immunoglobulin E-mediated allergy to drugs and substances used during general anaesthesia as well as non-allergic drug hypersensitivity reactions may account for anaesthesia-induced anaphylaxis. As IgE-mediated anaphylaxis is a potentially life-threatening reaction, identification of the culprit allergen is essential to avoid anaphylaxis recurrence during subsequent general anaesthesia. OBJECTIVE: To study whether preventive recommendations derived from allergy testing after intraoperative anaphylaxis were followed in subsequent general anaesthesia. METHODS: Results of standardized allergy testing after anaesthesia-induced anaphylaxis and outcome of subsequent general anaesthesia were analysed retrospectively. RESULTS: Fifty-three of 107 patients were diagnosed with IgE-mediated allergy to a drug or substance used during general anaesthesia, and 54 patients were test negative. Twenty-eight of 29 allergy patients tolerated subsequent general anaesthesia uneventfully. One patient with cefazolin allergy suffered from anaphylaxis recurrence due to accidental reapplication of cefazolin. Twenty-two of 24 test-negative patients tolerated subsequent general anaesthesia, whereas two patients again developed anaphylaxis despite pre-medication regimens. CONCLUSION AND CLINICAL RELEVANCE: Our results confirm the practical impact of allergy testing in general anaesthesia-induced anaphylaxis. By identification of the allergen, it is possible to avoid allergic anaphylaxis during subsequent anaesthesia. In most cases, recommended pre-medication seems to prevent the recurrence of non-allergic drug hypersensitivity reactions.

Pathogen-reduced Ebola virus convalescent plasma: first steps towards standardization of manufacturing and quality control including assessment of Ebola-specific neutralizing antibodies.

BACKGROUND: Ebola virus disease is a public health emergency of international concern, and enormous efforts are being made in the development of vaccines and therapies. Ebola virus convalescent plasma is a promising anti-infective treatment of Ebola virus disease. Therefore, we developed and implemented a pathogen-reduced Ebola virus convalescent plasma concept in accordance with national, European and global regulatory framework. MATERIALS AND METHODS: Ebola virus convalescent plasma manufacture and distribution was managed by a collection centre, two medical centres and an expert group from the European Blood Alliance. Ebola virus convalescent plasma was collected twice with an interval of 61 days from a donor recovering from Ebola virus disease in Germany. After pathogen reduction, the plasma was analysed for Ebola virus-specific immunoglobulin G (IgG) antibodies and its Ebola virus neutralizing activity. RESULTS: Convalescent plasma could be collected without adverse events. Anti-Ebola virus IgG titres and Ebola-specific neutralizing antibodies in convalescent plasma were only slightly reduced after pathogen reduction treatment with S59 amotosalen/UVA. A patient in Italy with Ebola virus disease was treated with convalescent plasma without apparent adverse effects. DISCUSSION: As proof of principle, we describe a concept and practical implementation of pathogen-reduced Ebola virus convalescent plasma manufacture, quality control and its clinical application to an Ebola virus disease patient.

Four amino acid exchanges located in the alpha-2 domain specify the novel HLA-B*50:20 allele.

HLA-B*50:20 contains seven nucleotide substitutions leading to four amino acid changes in the alpha-2 domain.

Determination of unacceptable HLA antigen mismatches in kidney transplant recipients: recommendations of the German Society for Immunogenetics.

One of the major tasks of histocompatibility and immunogenetics laboratories is the pretransplant determination of unacceptable antigen mismatches (UAM) in kidney transplant recipients. In this procedure, human leucocyte antigen (HLA) specificities are defined against which the patient has circulating alloantibodies that are expected to harm the transplanted organ. Using the information on UAM and the potential donor's complete HLA typing, prediction of the crossmatch result, the so called 'virtual crossmatch', is possible. Currently, the laboratories are using different algorithms for the determination of UAM, and depending on the algorithm, more or fewer organ offers are excluded for patients with a similar antibody profile. In order to bring homogeneity into the allocation of organs to immunized patients in Germany, the German Society for Immunogenetics established, on the basis of current knowledge, recommendations for the determination of UAM. The UAM recommendations, which are thought to serve as a common tool for responsible physicians at different transplant centers, contain technical issues that need to be considered and are individualized for sensitized patients with a high or intermediate risk of antibody-mediated rejection. The present review contains these recommendations and puts them into perspective to current international practice.

The novel alleles HLA-B*44:101 and HLA-B*57:48 of Caucasian origin are characterized by amino acid substitutions in the alpha 2 domain.

HLA-B*44:101 and HLA-B*57:48 are characterized by amino acid substitutions in the alpha 2 domain.

The novel HLA-C*12:92 allele is characterized by one amino acid exchange located in the T-cell receptor binding region of the alpha 2 domain.

HLA-C*12:92 contains one amino acid exchange in the T-cell receptor binding region.

Safety and efficacy of healthy volunteer stem cell mobilization with filgrastim G-CSF and mobilized stem cell apheresis: results of a prospective longitudinal 5-year follow-up study.

BACKGROUND AND OBJECTIVES: G-CSF-mobilized peripheral blood stem cells have long replaced marrow as the major source for allogeneic transplants. Conclusive evidence questioning the long-term safety of G-CSF for donors has not been provided, but the cumulative number of followed donors remains insufficient to rule out rare adverse events. A long-term active follow-up study of G-CSF-mobilized healthy volunteer donors was therefore performed. PATIENTS AND METHODS: Two hundred and three successive donors were evaluated pre-apheresis, subjected to G-CSF-mobilization/apheresis, and actively followed for 5 years by the same physicians and laboratories. Follow-up laboratory work included standard biochemical/haematological tests and T-cell phenotyping. RESULTS: Donor epidemiology was typical for reported stem cell donor cohorts. Acute adverse effects of G-CSF and apheresis were mild and transient, consistent with the previous reports. Mean circulating CD34(+) cells after nine doses of G-CSF were 124 per µl. Other biochemical/haematological parameters were also altered, consistent with G-CSF treatment. Spleen enlargement was modest. At first follow-up, all clinical and laboratory parameters had normalized. Leucocyte/lymphocyte counts and CD4/CD8 ratios were the same as during premobilization work-up and remained unchanged throughout. A single severe but likely unrelated adverse event, a case of papillary thyroid carcinoma, was reported. CONCLUSION: The studies add an observation time of almost 500 donor years to the growing body of evidence of the long-term safety of G-CSF for allogeneic donor stem cell mobilization.

Osseointegration of SLActive implants in diabetic pigs.

OBJECTIVES: Diabetes mellitus is currently classified as a relative contraindication for implant treatment because of microangiopathies with the consequence of impaired bone regeneration and higher rates of implant failure. The study aim was to investigate peri-implant bone formation in a diabetic animal model in comparison to healthy animals and to evaluate the differences between conventional (SLA(®) ) and modified (SLActive(®) ) titanium implant surfaces on osseointegration. MATERIAL AND METHODS: Each six implants were placed in the calvaria of 11 diabetic and 4 healthy domestic pigs. At 30 and 90 days after implant placement, the bone-to-implant contact (BIC) and bone density (BD) were appraised. Additionally, the expression of the bone-matrix proteins collagen type I and osteocalcin was evaluated at both points in time by using immunohistochemical staining methods. RESULTS: Overall, BIC was reduced in the diabetic group at 30 and 90 days. After 90 days, the SLActive(®) implants showed significantly higher BICs compared with the SLA(®) implants in diabetic animals. Peri-implant BD was higher in the SLActive(®) group at 30 and 90 days in healthy and diabetic animals. Collagen type I protein expression was higher using SLA(®) implants in diabetic pigs at 30 days. Values for osteocalcin expression were not consistent. CONCLUSIONS: The results indicate the negative effect of untreated diabetes mellitus on early osseointegration of dental implants. The modified SLA(®) surface (SLActive(®) ) elicited an accelerated osseointegration of dental implants, suggesting that a better prognosis for implant treatment of diabetic patients is possible.

16(th) IHIW: anti-HLA alloantibodies of the of IgA isotype in re-transplant candidates.

In this multicentre study, sera from 803 retransplant candidates, including 775 kidney transplant recipients, were analysed with regard to the presence and specificity of anti-HLA alloantibodies of the IgA isotype using a modified microsphere-based platform. Of the kidney recipients, nearly one-third (n = 237, 31%) had IgA alloantibodies. Mostly, these antibodies were found in sera that also harboured IgG alloantibodies that could be found in a total of 572 (74%) of patients. Interestingly, IgA anti-HLA antibodies were preferentially targeting HLA class I antigens in contrast to those of the IgG isotype, which targeted mostly both HLA class I and II antigens. Donor specificity of the IgA alloantibodies could be established for over half of the 237 patients with IgA alloantibodies (n = 124, 52%). A further 58 patients had specificities against HLA-C or HLA-DP, for which no information regarding donor typing was available. In summary, these data showed in a large cohort of retransplant candidates that IgA alloantibodies occur in about one-third of patients, about half of these antibodies being donor specific.

In vitro evaluation of the efficacy of commercial green tea extracts in UV protection.

Plants with antioxidant properties are beneficial for preventing the ageing events evoked by UV light, and numerous products based on Camellila sinensis (green tea) are commercially available, many of which claiming to contain bioactive compounds that would prevent UV-induced skin damage. In this study, we tested the efficacy of five commercial green tea extracts used to enrich cosmetic formulations for protecting human and mouse fibroblasts against UV radiation effects and compared with a fluid one prepared according to the Brazilian Pharmacopoeia recommendations. Taking into consideration that the ageing process can be accelerated by solar radiation by excessive free radical generation, leading to depletion of skin antioxidant defences, and its collapse caused by disruption of the metalloproteinase metabolism, we have used their individual (-)-epigallocathechin-3-gallate (EGCG) content, the catalase and SOD status and the matrix-degrading metalloproteases (MMP)-1, MMP-9 and MMP-13 levels as comparative parameters. The EGCG content of the commercial products showed wide variability, ranging from undetectable levels to 58.65 ± 1.12 µg mL(-1) , in contrast with the fluid extract (87.82 ± 1.35 µg mL(-1) ). Moreover, only the pharmacopoeic extract was able to significantly reduce MMP degradation while enhancing the levels of SOD and catalase. These results indicate, for the first time, that the methodologies for preparing herbal mixtures can interfere significantly with compounds endowed with photoprotective effects, and the efficacy of products containing C. sinensis extracts thought to act against effects of solar radiation can be compromised.

One amino acid change located in the conserved region of the alpha 1 domain specifies the novel HLA-C*07:147 allele.

The novel HLA-C allele HLA-C*07:147 contains one nucleotide substitution in exon 2 leading to an amino acid change in the alpha 1 domain from phenylalanine to leucine.

Two amino acid changes located in the alpha 1 domain specify the novel HLA-B*27:67 allele affecting the peptide-binding-site characteristics.

The novel HLA-B*27:67 contains three nucleotide substitutions in exon 2 leading to two amino acid changes.

The novel allele HLA-DQB1*0636 of Caucasian origin has a unique amino acid exchange at position 186 of the beta 2 region.

The novel HLA-DQB1*0636 allele differs from HLA-DQB1*060401 in one nucleotide substitution at codon 186 in exon 3.

A haplotype HLA-A*3201 - B*0702 - Cw*07 with a new HLA-C allelic variant closely related to HLA-Cw*0702 found in a Caucasian patient suffering from leukaemia.

The novel allele human leucocyte antigen (HLA)-Cw*0746 differs from HLA-Cw*070101 by three nucleotide exchanges at codons 45 and 66 in exon 2 and at codon 99 in exon 3.

Association of KIR2DL2 polymorphism rs2756923 with type 1 diabetes and preliminary evidence for lack of inhibition through HLA-C1 ligand binding.

Killer cell immunoglobulin-like receptors (KIRs) on chromosome 19q13.4 regulate the function of not only human natural killer (NK) cells but also T cells. An increase in activating KIR- human leucocyte antigen ligand pairs has been associated with an additional risk to develop type 1 diabetes (T1D). T1D families [n = 184 (552 individuals); n = 176 (528 subjects)], unrelated T1D patients (n = 380; n = 394) and healthy controls (n = 315; n = 401) from Germany and Belgium, respectively, were genotyped for the rs2756923 polymorphism within the KIR gene cluster haplotype B in exon 8 of the KIR2DL2 gene. We observed in both Germans and Belgians an overtransmission of the allele 'G' of the KIR2DL2-rs2756923 polymorphism (64.2% vs 35.8%, P = 3 x 10(-4) and 60.0% vs 40.0%, P = 0.02, respectively). In addition, this allele was more frequent in German patients than in healthy controls (78.4% vs 21.6%, P = 1 x 10(-3)). Preliminary results from a cytotoxicity assay suggest that inhibition of NK-cell cytotoxicity may be impaired in individuals carrying the rs2756923 G allele. These data suggest a potential role of the KIR2DL2-rs2756923 polymorphism in T1D in Germans and Belgians.

Mating in parents of type 1 diabetes families as a function of the HLA DR-DQ haplotype.

AIM: The Major Histocompatibility Complex (MHC) region on chromosome 6p21 (IDDM1) contributes about half of the familial clustering of type 1 diabetes (T1D). Several studies have revealed that highly polymorphic genes within the MHC may associate with the mating choice. Our study should determine whether a specific mating effect is detectable in T1D families as a function of human leucocyte antigen (HLA) DR-DQ, which could contribute to disease susceptibility. METHODS: We analysed the parental HLA-DR genotypes in 829 diabetic families. The families derive from the Type 1 Diabetes Genetics Consortium (T1DGC) in addition to those of our own centre and the original UK, US and SCAND diabetic families. RESULTS: A total of 307 of 829 parental couples (37.0%) were matched for at least one known T1D risk haplotype (DR3 or DR4), which is significantly less than the expected 374.9 (45.2%), derived from population genotype frequencies (p < 0.0009). Parents share less susceptibility haplotypes and rather complement each other as both carry one different risk haplotype (DR3 or DR4). The number of such parental couples was significantly higher than expected (293 vs. 223.4; p < 0.0003). All non-transmitted DR haplotype pairs were also analysed. More often than expected, both parents did not transmit DR1 (94 vs. 59.1; p < 0.003) and DRy (y: not DR1, not DR3, not DR4; 63 vs. 30.3; p < 0.0005). In contrast, the parental non-transmitted pair of haplotypes DR1-DRy was observed to a far lesser extent than expected (26 vs. 84.7; p < 10(-8)). These observations were only made in multiplex families, whereas in simplex families, no deviation from the expected frequencies was observed. CONCLUSIONS: Our data are consistent with the conclusion that genes in the HLA region may influence the mating choice in parents of T1D patients, thus contributing to familial clustering of T1D in multiplex families. This may indicate a different parental background of multiplex compared with simplex T1D families.