Andrea Marchesi

A new automated cell washer device for thawed cord blood units.

BACKGROUND:  The current available techniques to wash out DMSO from thawed umbilical cord blood (UCB) units are based essentially on standard centrifugation in an open system with various degrees of cell loss.

The plasma levels of soluble HLA-G molecules correlate directly with CD34+ cell concentration and HLA-G 14bp insertion/insertion polymorphism in cord blood donors.

BACKGROUND Cord blood provides haematopoietic stem cells for allogeneic transplantation and, thanks to the naivety of its immune system, has several advantages over other sources of stem cells. In the transplantation setting, the presence of immunosuppressive human leucocyte antigen (HLA)-G molecules has been advocated to prevent both rejection and Graft-versus-Host disease. HLA-G is physiologically expressed throughout pregnancy and is contained in cord blood at birth. Moreover, it has recently been reported that not only cord blood mesenchymal cells, but also CD34+ cell progenies produce soluble HLA-G (sHLA-G). We tried to identify the largest producer of sHLA-G among 85 healthy cord blood donors at Pavia Cord Blood Bank, correlating the sHLA-G concentration with the HLA-G 14bp insertion/deletion (INS/DEL) genotype and CD34+ cell concentration. MATERIALS AND METHODS We measured sHLA-G levels in 36 cord blood plasma stored at -20 °C for 2 months and 49 cord blood plasma stored at -196 °C for 4-6 years, by enzyme-linked immunosorbent assay. All cord blood donors were genotyped for the HLA-G 14bp INS/DEL polymorphism by polymerase chain reaction. For each cord blood unit, we measured the cell concentration by flow cytometry. RESULTS We did not find differences in sHLA-G levels between cord blood plasma aliquots stored for 4-6 years at -196 °C and cord blood plasma aliquots stored for 2 months at -20 °C. We observed a higher sHLA-G concentration in cord blood plasma donors who carried the HLA-G 14bp INS/INS genotype and had higher CD34+ cell concentrations (P=0.006). DISCUSSION This is the first report showing that the best cord blood stem cell donor is also the best sHLA-G producer, particularly if genetically characterized by the HLA-G 14bp INS/INS genotype. If the therapeutic role of sHLA-G molecules were to be finally established in the transplantation setting, our data suggest that cord blood plasma donors can provide a safe source of allogeneic sHLA-G immunosuppressive molecules ready for transfusion.

Intragenomic conflict of maternal HLA haplotypes: A potential link between vigorous intrauterine growth and risk of autoimmunity in adulthood

The haplotype segregation rate ofMendelian heredity is ordinarily taken for granted. Nevertheless, there are numerous examples of outsider genes that preferentially perpetuate themselves in the population by biasing the Mendelian process; this mechanism is known as intragenomic conflict. A clear mechanism of intragenomic conflict operates in the human leukocyte antigen (HLA) locus, HLA-DR, favoring the transmission of HLADR3 and HLA-DR4 alleles, even though both predispose an individual to diabetes (Noble and Valdes, 2011). In diabetic families, an unexpectedly high proportion (>50%) of newborns carry HLA-DR3 or HLA-DR4 allele, and conversely a disproportionate number of fetuses with an allele other than HLA-DR3 or HLA-DR4 appear to have died in utero. Thus, the inheritance of HLA-DR3 and HLA-DR4 alleles is undoubtedly favorable for intrauterine growth, despite predisposing the carriers to developing diabetes during their lifetime. We investigated the presence of intragenomic conflict in HLA maternal transmission by examining the HLA two-point and three-point haplotypes (marked by HLA-A, HLA-B, and HLA-DRB1 polymorphisms) of 1,206 infant cord blood donors and their corresponding mothers enrolled to the Pavia Cord Blood Bank, following the FACT Netcord international standards (Suppement 1). HLA-A, HLA-B, and HLA-DRB1 polymorphisms were analyzed by PCR techniques, and each genotyping was unambiguously assessed following the European Federation of Immunogenetics recommendations. Fetal HLAhaplotypeswere assignedbymaternal segregation. We found several two-point and three-point, preferentially inherited (PI) and non-inherited (PnI) HLA haplotypes from the maternal side (Table 1). As our sample includes only healthy neonates born from physiological pregnancies (Suppement 1), we conclude that the HLA PI haplotypes are favorable for intrauterine growth, whereas the HLA PnI haplotypes are disadvantageous. In the literature, the majority of the HLA PI haplotypes correlate with adult autoimmune conditions, whereas only two HLA PnI haplotypes correlate with infant autoimmune conditions (Table 1). Further, wepreviously observed a correlation between fetal growth and specificHLA haplotypes connected to the risk of developing autoimmune conditions: the Ancestral Haplotype AH8.1 (HLA-A*01;B*08;DRB1*03) is overrepresented in high-weighted babies (Capittini et al., 2009) despite its association with a wide range of immune-mediated pathologies in adulthood (Price et al., 1999). Theantagonistic pleiotropy theory suggests that reproductionhasacost in longevity: a genetic variation that favors higher fitness will be advantaged in future generations, even if this variation has possible deleterious effects during lifetime. Our observations support the hypothesis that certain HLA haplotypes with initial beneficial effects (improve fetal survival in uterus) prevail, despite their negative effects on adult health (increase the risk of developing autoimmune diseases). In particular, our healthy cord blood donors have preferentially inherited a typology of maternal HLA haplotypes associated with adult autoimmune diseases (Table 1) that present beyond the fertile age, and thus have no influence on fecundity. The preferential inheritance of specific HLA haplotypes (like HLA-DR3 and HLA-DR4 alleles in diabetic families) represents a clear instance of an evolutionary-balanced risk for fetuses, that is a reduced risk of miscarriage in return for a higher risk of developing an autoimmune disease. This concept of fetal protection-at-a-price may help us decipher an evolutionary context to autoimmune diseases. Yet, wemust still understand the immune mechanisms balancing vigorous intrauterine growth (where the mother tolerates the fetal allograft) with adult autoimmunity (where the immune system lost the self-recognition).