► Mesenchymal stem cells were grown in a novel hollow-fiber based bioreactor system. ► We show preclinical large-scale animal-free expansion of MSC in a GMP-compliant manner. ► We show ...immunosuppressive and biologic properties of novel bioreactor-expanded MSC.
Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials.
We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15–27days. 2–58million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13days led to further 10–20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare condition in which maternal alloantibodies to fetal platelets cause fetal thrombocytopenia that may lead to intracranial hemorrhage ...(ICH). Off-label intravenous immunoglobulin (IVIg) has for 30 years been the standard of care for pregnant women who previously have had a child with FNAIT. The efficacy of this treatment has never been tested in a placebo-controlled clinical trial. Although IVIg treatment may improve the neonatal outcome in women who previously have had a child with FNAIT-associated ICH, the question is whether IVIg is necessary for all immunized pregnant women at risk of having a child with FNAIT. The results from some recent publications suggest that antenatal IVIg treatment is not necessary for women who are (1) HPA-1a-immunized and HLA-DRB3*01:01-negative, (2) HPA-1a-immunized with a previous child with FNAIT but without ICH or (3) HPA-5b-immunized. If IVIg is not used for these categories of pregnant women, the amount of IVIg used in pregnant women with platelet antibodies would be reduced to less than ¼ of today’s use. This is important because IVIg is a scarce resource, and the collection of plasma for the treatment of one pregnant woman is not only extremely expensive but also requires tremendous donor efforts.
All non-sensitized Rhesus D (RhD)-negative pregnant women in Germany receive antenatal anti-D prophylaxis without knowledge of fetal RhD status. Non-invasive prenatal testing (NIPT) of cell-free ...fetal DNA in maternal plasma could avoid unnecessary anti-D administration. In this paper, we systematically reviewed the evidence on the benefit of NIPT for fetal RhD status in RhD-negative pregnant women.
We systematically searched several bibliographic databases, trial registries, and other sources (up to October 2019) for controlled intervention studies investigating NIPT for fetal RhD versus conventional anti-D prophylaxis. The focus was on the impact on fetal and maternal morbidity. We primarily considered direct evidence (from randomized controlled trials) or if unavailable, linked evidence (from diagnostic accuracy studies and from controlled intervention studies investigating the administration or withholding of anti-D prophylaxis). The results of diagnostic accuracy studies were pooled in bivariate meta-analyses.
Neither direct evidence nor sufficient data for linked evidence were identified. Meta-analysis of data from about 60,000 participants showed high sensitivity (99.9%; 95% CI 99.5%; 100% and specificity (99.2%; 95% CI 98.5%; 99.5%).
NIPT for fetal RhD status is equivalent to conventional serologic testing using the newborn's blood. Studies investigating patient-relevant outcomes are still lacking.
CD177 is a glycosyl phosphatidyl inositol (GPI)-linked, neutrophil-specific glycoprotein that in 3-5% of normal individuals is absent from all neutrophils. The molecular mechanism behind the absence ...of CD177 has not been unravelled completely. Here, we analyse the impact of the recently described
c.1291G>A variant on CD177 expression. Recombinant
c.1291G>A was expressed in HEK293F cells and its expression on the cell surface, inside the cell, and in the culture supernatant was investigated. The
c.1291G>A protein was characterised serologically and its interaction with proteinase 3 (PR3) was demonstrated by confocal laser scanning microscopy. Our experiments show that
c.1291G>A does not interfere with CD177 protein biosynthesis but affects the membrane expression of CD177, leading to very low copy numbers of the protein on the cellular surface. The mutation does not interfere with the ability of the protein to bind PR3 or human polyclonal antibodies against wild-type CD177. Carriers of the c.1291G>A allele are supposed to be phenotyped as CD177-negative, but the protein is present in soluble form. The presence of
c.1291A leads to the production of an unstable CD177 protein and an apparent "CD177-null" phenotype.
Platelet autoantibody-induced platelet clearance represents a major pathomechanism in immune thrombocytopenia (ITP). There is growing evidence for clinical differences between anti-glycoprotein ...IIb/IIIa and anti-glycoprotein Ib/IX mediated ITP. Glycoprotein V is a well characterized target antigen in Varicella-associated and drug-induced thrombocytopenia. We conducted a systematic study assessing the prevalence and functional capacity of autoantibodies against glycoprotein V. A total of 1140 patients were included. In one-third of patients, platelet-bound autoantibodies against glycoproteins Ib/IX, IIb/IIIa, or V were detected in a monoclonal antibody immobilization of platelet antigen assay; platelet-bound autoantiglycoprotein V was present in the majority of samples (222 out of 343, 64.7%). Investigation of patient sera revealed the presence of free autoantibodies against glycoprotein V in 13.5% of these patients by an indirect monoclonal antibody immobilization of platelet antigen assay, but in 39.6% by surface plasmon resonance technology. These antibodies showed significantly lower avidity (association/dissociation ratio 0.32±0.13
0.73±0.14;
<0.001). High- and low-avidity antibodies induced comparable amounts of platelet uptake in a phagocytosis assay using CD14
positively-selected human macrophages mean phagocytic index, 6.81 (range, 4.75-9.86)
6.01 (range, 5.00-6.98);
=0.954. In a NOD/SCID mouse model, IgG prepared from both types of anti-glycoprotein V autoantibodies eliminated human platelets with no detectable difference between the groups from the murine circulation mean platelet survival at 300 minutes, 40% (range, 27-55)
35% (16-46);
=0.025. Our data establish glycoprotein V as a relevant immune target in immune thrombocytopenia. We would suggest that further studies including glycoprotein V will be required before ITP treatment can be tailored according to platelet autoantibody specificity.
BACKGROUND
Neutrophil specific Fcγ receptor IIIb (CD16b) is a low‐affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA‐1a and HNA‐1b differ in four ...amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA‐1a, −1b epitopes is currently unknown.
STUDY DESIGN AND METHODS
Permutation of each polymorphic amino acid from wild‐type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well‐characterized HNA antisera in an antigen capture assay.
RESULTS
Analyzing the reaction pattern revealed that anti‐HNA‐1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti‐HNA‐1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti‐HNA‐1a and anti‐HNA‐1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind.
CONCLUSION
Whereas the primary structure of HNA‐1a and HNA‐1b usually differs in four amino acids, epitope composition is not “antithetical”. N65 alone determines the presence of HNA‐1a, and S36 and/or N82 determine the presence of HNA‐1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA‐1 phenotype is predicted from a genotype.
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads ...to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14
CD16
), intermediate (CD14
CD16
), and nonclassical (CD14
CD16
) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14
CD16
), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin-semaphoring-integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable
platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis
and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future.
BACKGROUND
Fetal human platelet antigen (HPA) genotyping is required to determine whether the fetus is at risk and whether prenatal interventions to prevent fetal bleeding are required in pregnant ...women with a history of fetal and neonatal alloimmune thrombocytopenia (FNAIT). Methods for noninvasive genotyping of HPA alleles with the use of maternal plasma cell‐free DNA were published recently but do lack internal controls to exclude false‐negative results.
STUDY DESIGN AND METHODS
Cell‐free DNA was isolated from plasma of four pregnant women with a history of FNAIT caused by anti‐HPA‐1a and controls. A primer panel was designed to target sequences flanking single‐nucleotide polymorphisms (SNPs)/exonic regions of ITGB3 (HPA‐1), ITGA2B (HPA‐3), ITGA2 (HPA‐5), CD109 (HPA‐15), RHD, RHCE, KEL, DARC, SLC14A1, GYPA, GYPB, and SRY. These regions and eight anonymous SNPs were massively parallel sequenced by semiconductor technology.
RESULTS
The mean (±SD) number of reads for targeted SNPs was 5255 (±2838). Fetal DNA was detected at a median of 4.5 (range, 2‐8) polymorphic loci. The mean fractional fetal DNA concentration in cell‐free maternal plasma was 8.36% (range, 4.79%‐15.9%). For HPA‐1, nonmaternal ITGB3 sequences (c.176T, HPA‐1a) were detected in all HPA‐1ab fetuses. One HPA‐1bb fetus was unequivocally identified, showing the pregnancy was not at risk of FNAIT.
CONCLUSION
We have successfully established massively parallel sequencing as a novel reliable method for noninvasive genotyping of fetal HPA‐1a alleles. This technique may also allow the safe detection of other fetal blood group polymorphisms frequently involved in FNAIT and hemolytic disease of the newborn.
BACKGROUND
An alloimmune response to red blood cell (RBC) transfusion in neonates is a rare event. Several guidelines recommend limited pretransfusion testing in neonates. The evidence for these ...recommendations is based on small studies with sample sizes of between 30 and 90 infants.
STUDY DESIGN AND METHODS
We conducted a retrospective cohort study among consecutive patients who received transfusions at a single university medical center. All non‐alloimmunized patients who had received their first RBC transfusion between 1994 and 2013 and who underwent at least one antibody screening follow‐up visit between 7 and 365 days after transfusion were included.
RESULTS
The incidence of alloimmunization in the control group of 17,084 adult patients age 45 years or older who had received a median of 5 RBC units (interquartile range, 2‐12 RBC units) was 3.55% (n = 607 alloimmunized patients). After transfusion of 40 RBC units, the cumulative incidence of alloimmunization in adult controls was 10.24% (95% confidence interval, 7.71%‐13.17%). In total, 1641 neonates and children up to age 3 years received a median of 4 RBC units (interquartile range, 2‐7 RBC units) in a median of two RBC transfusion episodes (interquartile range, one to five RBC transfusion episodes). Two children developed anti‐M and anti‐E antibodies post‐transfusion at the ages of 181 and 611 days, respectively.
CONCLUSION
To our knowledge, this study presents the largest longitudinal cohort study of RBC alloimmunization in neonates. Antibodies against RBC antigens were not detected within the first 6 months of life. Repeat antibody screening and cross‐matching during the first months of life can be safely omitted.
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