Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources. cell separation methods/fluorescence in-situ hybridization/hereditary diseases/polymerase chain reaction/pregnancy     

Utilization patterns of frozen autologous red blood cells. Experience in a referral center and a community hospital

The risks of homologous blood transfusion have motivated some blood centers and private industry to consider providing long-term storage of frozen, autologous red blood cells as a service. The usefulness of this practice is unknown. We performed a retrospective analysis of frozen autologous red blood cell use in two hospitals. Records were available for 21- and 9-year intervals, respectively. A total of 104 autologous units were cryopreserved for 41 patients. Fifteen (37%) of 41 patients received one or more of their stored units of red blood cells. Twenty-two patients had autologous units frozen in anticipation of elective surgery; 11 (50%) of these 22 patients received some or all of their stored units. Sixteen patients had autologous units stored because of potential transfusion problems related to rare blood types or to the presence of multiple blood cell alloantibodies, and another 3 patients had units frozen simply at their personal request. Only 4 (21%) of these latter 19 patients who donated without a specific planned use eventually received their frozen autologous red blood cells. Long-term autologous frozen red blood cell storage can improve medical management of some patients with anticipated surgical procedures or unusual requirements for transfusion. However, our study suggests that most autologous units frozen without specific planned use will not be transfused.     

The p150,95 molecule is a marker of human mononuclear phagocytes: comparison with expression of class II molecules

A new monoclonal antibody (mAb), named 3.9, is described that is specific for the p150,95 molecule, a member of the LFA-1, CR3, p150,95 family of human leukocyte differentiation antigens. The LFA-1 molecule participates in a variety of T cell interactions and the CR3 molecule is the receptor for the complement component iC3b, but little is known about the p150,95 molecule. Here we show that the expression of p150,95 is confined to myeloid cells. mAb 3.9 reacts variably with neutrophils, more strongly with monocytes and is most strongly expressed on tissue macrophages. Using this mAb and others, we have examined the heterogeneity of tissue macrophages. Cells such as Langerhans' cells, dendritic reticulum cells and osteoclasts failed to react with these mAb and thus, probably do not belong to the mononuclear phagocyte lineage. Using a new double-labeling technique, we investigated lymphoid tissue for dendritic cells bearing class II molecules which might function in interactions with T cells. In T cell areas macrophages expressing class II markers were seen but there was no evidence for other types of dendritic or interdigitating cells which expressed class II molecules but not macrophage epitopes. The conclusion from this survey was that the most prominent cell with dendritic morphology found in the T cell areas of lymphoid tissue was a macrophage.     

Tales of tails: regulation of telomere length and telomerase activity during lymphocyte development, differentiation, activation, and aging

Summary: Telomerase activity and the regulation of telomere length are factors which have been implicated in the control of cellular replication. These variables have been examined during human lymphocyte development, differentiation, activation, and aging. It was found that telomere length of peripheral blood CD4⁺ T cells decreases with age as well as with differentiation from naive to memory cells in vivo , and decreases with cell division in vitro. These results provide evidence that telomere length correlates with lymphocyte replicative history and residual replicative potential. In contrast, telomere length appears to increase during tonsil B-cell differentiation and germinal center (GC) formation in vivo. It was also found that telomerase activity is highly regulated during T-cell development and B-cell differentiation in vivo , with high levels of telomerase activity expressed in thymocytes and GC B cells, and low levels of telomerase activity in resting mature peripheral blood lymphocytes. Finally, resting lymphocytes retain the ability to upregulate telomerase activity upon activation, and this capacity does not appear to decline with age. Although the precise role of telomerase in lymphocyte function remains to be elucidated, telomerase may contribute to protection from telomere shortening in T and B lymphocytes, and may thus play a critical role in lymphocyte development, differentiation and activation. The future study of study telomerase and its regulation of telomere length may enhance our understanding of bow the replicative lifespan is regulated in lymphocytes.     

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.     

A chimeric T cell receptor with super-signaling properties

A key question yet to be resolved concerns the structure and function relationship of the TCR complex. How does antigen recognition by the TCR-alphabeta chains result in the activation of distinct signal transduction pathways by the CD3-gammadeltaepsilon/zeta complex? To investigate which part of the TCR-beta chain is involved in TCR signaling, we exchanged different domains of the constant regions of the TCR-beta chain with the corresponding TCR-gamma chain domains. We show here that hybridoma cells expressing a chimeric TCR-beta chain (betaIII) containing intracellular and transmembrane TCR-gamma amino acids, together with a wild-type TCR-alpha (alphawt) chain, were 10 times more sensitive to antigenic stimulation compared to cells expressing TCR-alphawt/betawt chains. This super-signaling phenotype of the betaIII chain was observed in two different TCRs. One specific for an alloantigen (I-A(bm12)) and one for an autoantigen (I-A(b)/MOG(35-55)). We found that this chimeric alphawt/betaIII TCR had normal association with CD3-gammadeltaepsilon and zeta chains. To investigate the effect of the chimeric betaIII chain in transgenic T cells, we made MOG(35-55)-specific TCR transgenic mice expressing either the alphawt/betawt or chimeric alphawt/betaIII TCR. Similar to what was observed in hybridoma cells, transgenic alphawt/betaIII T cells showed a super-signaling phenotype upon antigenic stimulation. Further studies may help us understand the effect of increased TCR signaling on autoimmunity and may lead to the identification of signaling molecules that can be targeted to stop the progression of autoimmune disorders such as multiple sclerosis.     

Location,identity, amount and serial entry of chloroplast DNA sequences in crucifer mitochondrial DNAs

Southern blot hybridization techniques were used to examine the chloroplast DNA (cpDNA) sequences present in the mitochondrial DNAs (mtDNAs) of two Brassica species (B. campestris and B. hirta), two closely related species belonging to the same tribe as Brassica (Raphanus sativa, Crambe abyssinica), and two more distantly related species of crucifers (Arabidopsis thaliana, Capsella bursa-pastoris). The two Brassica species and R. sativa contain roughly equal amounts (12–14 kb) of cpDNA sequences integrated within their 208–242 kb mtDNAs. Furthermore, the 11 identified regions of transferred DNA, which include the 5 end of the chloroplast psaA gene and the central segment of rpoB, have the same mtDNA locations in these three species. Crambe abyssinica mtDNA has the same complement of cpDNA sequences, plus an additional major region of cpDNA sequence similarity which includes the 16S rRNA gene. Therefore, except for the more recently arrived 16S rRNA gene, all of these cpDNA sequences appear to have entered the mitochondrial genome in the common ancestor of these three genera. The mitochondrial genomes of A. thaliana and Capsella bursa-pastoris contain significantly less cpDNA (5–7 kb) than the four other mtDNAs. However, certain cpDNA sequences, including the central portion of the rbcL gene and the 3 end of the psaA gene, are shared by all six crucifer mtDNAs and appear to have been transferred in a common ancestor of the crucifer family over 30 million years ago. 1n conclusion, DNA has been transferred sequentially from the chloroplast to the mitochondrion during crucifer evolution and these cpDNA sequences can persist in the mitochondrial genome over long periods of evolutionary time.     

Toll-like receptor 6 gene (TLR6): single-nucleotide polymorphism frequencies and preliminary association with the diagnosis of asthma

Toll-like receptor 6 (TLR6) is one of a series of highly conserved innate immune receptors. We resequenced TLR6 in DNA samples from 24 African Americans, 23 European Americans, and 24 Hispanic Americans, identifying 53 SNPs, 22 with an allele frequency >5%. Significant differences in SNP frequencies among the three populations were noted. In all, 11 SNPs caused amino-acid changes, including one with a frequency >5% in all three populations. Utilizing this SNP (Ser249Pro), we performed exploratory nested case-control disease-association studies, including one involving 56 African Americans with asthma and 93 African American controls. The minor allele of this SNP was associated with decreased risk for asthma (odds ratio 0.38, 95% CI 0.16-0.87, P=0.01), an effect consistent with the known biology of the toll-like receptors. Although replication of this finding in other, larger samples is needed, variation in TLR6 may have relevance to the pathogenesis of immunologically mediated diseases.