Assessment of differences in HLA-A, -B, and -DRB1 allele mismatches among African-American and non-African-American recipients of deceased kidney transplants.

Among recipients of deceased donor kidney transplants, African-Americans experience a more rapid rate of kidney allograft loss than non-African-Americans. The purpose of this study was to characterize and quantify the HLA-A, -B, and -DRB1 allele mismatches and amino acid substitutions at antigen recognition sites among African-American and non-African-American recipients of deceased donor kidney transplants matched at the antigen level. In recipients with zero HLA antigen mismatches, the degree of one or two HLA allele mismatches for both racial groups combined was 47%, 29%, and 11% at HLA-DRB1, HLA-B, and HLA-A, respectively. There was a greater number of allele mismatches in African-Americans than non-African-Americans at HLA-A (P < .0001), -B (P = .096), and -DRB1 loci (P < .0001). For both racial groups, the HLA allele mismatches were predominantly at A2 for HLA-A; B35 and B44 for HLA-B; but multiple specificities for HLA-DRB1. The observed amino acid mismatches were concentrated at a few functional positions in the antigen binding site of HLA-A and -B and -DRB1 molecules. Future studies are ongoing to assess the impact of these HLA mismatches on kidney allograft loss.

Expression of AML1-ETO in human myelomonocytic cells selectively inhibits granulocytic differentiation and promotes their self-renewal.

The t(8;21) translocation is one of the most frequent translocations in acute myeloid leukaemia (AML), giving rise to the AML1-ETO fusion protein (or RUNX1-CBF2T1). This abnormality is associated with myelocytic leukaemia with dysplastic granulopoiesis. Here, we demonstrate that when expressed in a normal human (CD34(+)) progenitor population, AML1-ETO selectively inhibits granulocyte colony formation but not monocyte colony formation. In bulk liquid culture, we found that though AML1-ETO transiently inhibited the proliferation of CD34(+) cells, it promoted long-term growth of myeloid cells for more than 80 days, suggesting that differentiation was inhibited. In support of this, cultures expressing AML1-ETO demonstrated enhanced retention of colony-forming capacity. Phenotypic examination of AML1-ETO cultures revealed a defect in granulocytic differentiation in terms of retention of CD34(+) cells within the culture and delayed CD11b upregulation. Morphologically, granulocyte terminal differentiation in AML1-ETO-expressing cells was inhibited by 83+/-5%, giving rise to a build-up of early to intermediate granulocytes that exhibited a number of morphological features associated with t(8;21) leukaemias. In contrast, AML1-ETO had little or no effect on monocytic differentiation. Taken together, these results suggest that expression of AML1-ETO selectively inhibits the differentiation of granulocytic cells and promoted extensive self-renewal, supporting a causal role for t(8;21) translocations in leukaemogenesis.

Estimation of mutation rate at human glycophorin A locus in hematopoietic stem cell progenitors.

Surveys of human mutant cells exhibit a few individuals with relatively high "outlying" values, which might be explained by rare mutations occurring during development. To estimate how commonly this occurs, mutant red cell frequencies at the glycophorin A locus in 135 neonates and 109 children and adolescents from three research centers are compared with simulations in which mutations arise from successive cycles of binary fission. The simulations predict the data most accurately when the mutation rate in stem cell precursors is about 2-4 x 10(-7) per division cycle, which is similar to previous estimates from adult stem cell divisions. If these mutation rates are accurate, and the number of stem cell divisions during adult life is as low as previously estimated, it is predicted that up to one-sixth of mutant stem cells over a lifetime arose in early life. However, these mutant stem cells would be difficult to detect in surveys because their distribution within the general population is so skewed.

Bcl-2 antisense oligonucleotides enhance the cytotoxicity of chlorambucil in B-cell chronic lymphocytic leukaemia cells.

We have previously shown that the Bcl-2 antisense oligonucleotide ODN 2009 can induce apoptosis in B-cell chronic lymphocytic leukaemia (B-CLL) cells. In this study we evaluated whether ODN 2009 could increase the sensitivity of B-CLL cells to Chlorambucil-induced cell death in vitro in order to establish whether the notion of antisense-mediated chemosensitisation could be applied to B-CLL. Bcl-2 antisense in combination with Chlorambucil resulted in a more marked reduction in Bcl-2 protein expression (p = 0.003), enhanced Bax expression (p < 0.0001) and increased apoptosis when compared to cells incubated with Chlorambucil alone (p = 0.03). This increased in vitro cytotoxicity demonstrates a proof of the concept that a combination of Bcl-2 antisense oligonucleotides with conventional chemotherapeutic drugs may elicit an enhanced therapeutic effect in B-CLL and should therefore be considered for further investigation in the form of a clinical trial.

Flavopiridol circumvents Bcl-2 family mediated inhibition of apoptosis and drug resistance in B-cell chronic lymphocytic leukaemia.

Flavopiridol, a synthetic flavone, is currently under clinical investigation for the treatment of B-cell chronic lymphocytic leukaemia (B-CLL). In this study, we examined the in vitro effects of flavopiridol and fludarabine on B-CLL cells from 64 patients (36 treated and 28 untreated) in terms of apoptosis induction and Bcl-2 family expression. Both flavopiridol and fludarabine induced apoptosis in all the samples tested with mean LD(50) values (+/- SD) of 59.7 nmol/l (+/- 36.5) and 6.2 micromol/l (+/- 7.5) respectively. Mean flavopiridol LD(50) values were not significantly different between the treated and untreated patient groups (P = 0.35), whereas the fludarabine LD(50) values were significantly higher in the previously treated patient group (P = 0.01). Bcl-2 and Mcl-1 expression were downregulated in both flavopiridol and fludarabine-induced apoptotic cells, but the increase in Bax expression that accompanied fludarabine-induced apoptosis was not evident in flavopiridol-treated cells. In addition, Bcl-2:Bax ratios were not predictive of flavopiridol cytotoxicity (P = 0.82), whereas they were highly predictive of in vitro responsiveness to fludarabine (P = 0.001). Overall, these findings suggest that flavopiridol exerts its cytotoxic effect through a novel cell-death pathway that is not subject to the Bcl-2 family mediated resistance mechanisms that reduce the efficacy of many conventional chemotherapeutic drugs.

Erythrocyte agglutination by wheat germ agglutinin: ionic strength dependence of the contact seam topology.

The topology of the cell-cell contact seam formed when normal or pronase pre-treated (PPT) erythrocytes are exposed to wheat germ agglutinin (WGA) in isotonic media of different ionic strengths was examined here. Lectin uptake and cell agglutination were also quantified. Agglutination of normal cells was gradually and significantly inhibited as ionic strength (IS) was reduced from 0.15 (buffered 145 mm NaCl) to 0.105. Agglutination was less inhibited in PPT cells, even when IS was reduced to 0.09. Cell contact seams formed during agglutination showed patterns of localized contacts. The scale of the patterns, i.e. the average lateral separation distance of contact regions, was 0.62 microm for normal cells and was significantly shorter, at 0.44 microm, for PPT cells at an IS of 0.15. The scale increased significantly for both cell types when the IS was reduced to 0.09. Flow cytometry measurements showed that WGA uptake by normal cells increased slightly, whilst that for PPT cells was unchanged, as IS was decreased from 0.15 to 0.09. The results imply that, whilst ionic strength change does not exert a strong influence on intermolecular WGA-ligand binding, physico-chemical modification of the interaction between cells modulates not only the extent and progression of the biospecific lectin-induced cell-cell agglutination but also the topology of the contact seam. The IS dependence of contact separation in WGA-agglutinated cells is contrasted here with that reported for cells adhering in dextran solutions. The influence of IS change and pronase pre-treatment on contact pattern are consistent with predictions, from interfacial instability theory, of punctuate thinning of the aqueous layer separating bilayer membranes in close apposition.

Late-onset erythropoietic porphyria caused by a chromosome 18q deletion in erythroid cells.

The erythropoietic porphyrias, erythropoietic protoporphyria and congenital erythropoietic porphyria, result from germline mutations in the ferrochelatase gene and uroporphyrinogen III synthase gene, respectively. Both conditions normally present in childhood but rare cases with onset past the age of 40 y have been reported. Here we show that late-onset erythropoietic protoporphyria can be caused by deletion of the ferrochelatase gene in hematopoietic cells with clonal expansion as part of the myelodysplastic process. This is the first direct demonstration of porphyria produced by an acquired molecular defect restricted to one tissue. Some other cases of late-onset erythropoietic porphyria may be explained by a similar mechanism.

Determination of red cell volume in infants needing blood transfusion.

The total circulating red cell volume (RCV) is a better guide to the oxygen-carrying capacity of the blood in the whole circulation than is the haemoglobin concentration (Hb) or haematocrit in a blood sample. Pre- and post-transfusion RCV (and blood volume (BV)) may be determined by flow cytometry by exploiting antigen differences between transfused donor red cells and the recipient's red cells. This paper describes the use of red cell antigen differences of Duffy, Kidd, MN and RhD between donor and recipient. In 20 infants, transfused on 21 occasions, pretransfusion RCV ranged from 12 to 39 mL kg(-1) body weight. Only at one transfusion could no usable donor-recipient antigen differences be exploited. Measurement of RCV, used routinely, may determine the transfusion requirements of sick infants more accurately, with the aim of normalizing RCV and BV--securing euvolaemia--at the end of the transfusion. This may allow a complete correction of the RCV deficiency at the first occasion of transfusion. This approach may reduce donor exposures and also optimize oxygen transport and organ perfusion of the infant undergoing intensive management, perhaps leading ultimately to improved survival rates and fewer long-term complications of neonatal intensive care.

Bcl-2 and bax expression and chlorambucil-induced apoptosis in the T-cells and leukaemic B-cells of untreated B-cell chronic lymphocytic leukaemia patients.

Chlorambucil and other cytotoxic drugs kill cells, non-selectively, by inducing apoptosis. In this study, we measured the apoptotic response to chlorambucil in T- and B-cells from untreated B-CLL patients and T-cells, from normal control subjects. We found increased chemosensitivity in the T-cells of B-CLL patients compared to the controls (P=0.0002). The chlorambucil ID(50) values for T-cells from B-CLL patients showed a direct correlation with Bcl-2 expression (P=0.002) and an inverse correlation with CD3 cell count (P<0.0001), suggesting a trend of increasing chemosensitivity and decreasing Bcl-2 expression with an elevated T-cell count. There was no differential expression of Bcl-2 or Bax between the CD4(+) and CD8(+) cells of B-CLL patients, isolated by immunomagnetic separation. We found correlations in the leukaemic B-cells between chlorambucil ID(50) values and both Bcl-2 expression (P=0.006), and Bcl-2/Bax ratios (P=0.002), suggesting a role for the Bcl-2/Bax ratio in predicting the response of untreated CLL patients to cytotoxic treatment. Chlorambucil produced almost identical changes in Bcl-2 and Bax expression in normal T-cells and leukaemic B-cells triggered to die by apoptosis, which together with the correlation between Bcl-2 and chemosensitivity confirms a pivotal role for Bcl-2 in regulating a distal step in the apoptotic pathway following cytotoxic cellular damage.

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy.

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.

Intercellular communication in the immune system: differential expression of connexin40 and 43, and perturbation of gap junction channel functions in peripheral blood and tonsil human lymphocyte subpopulations.

The distribution and function of connexins (integral membrane proteins assembled into gap junction intercellular communication channels) were studied in human lymphocyte subpopulations. The expression of mRNA encoding connexins in peripheral blood and tonsil-derived T, B and natural killer (NK) lymphocytes was examined. Connexin43 (Cx43) mRNA was expressed in peripheral blood and tonsil lymphocytes, but Cx40 mRNA expression was confined to tonsil-derived T and B lymphocytes; Cx26, Cx32, Cx37 and Cx45 were not detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis also demonstrated the presence of Cx40 and Cx43 proteins in T and B lymphocytes in a manner coincidental to the mRNA detection. Stimulation in vitro of T and B lymphocytes with phytohaemagglutinin (PHA) and lipopolysaccharide (LPS), respectively, increased Cx40 and Cx43 protein expression. Flow cytometric analysis, using antibodies to extracellular loop amino acid sequences of connexins, confirmed the surface expression of connexins in all lymphocyte subpopulations. Assembly of connexins into gap junctions providing direct intercellular channels linking attached lymphocytes was demonstrated by using a dye transfer technique. The exchange of dye between lymphocytes was inhibited by a connexin extracellular loop mimetic peptide and alpha-glycyrrhetinic acid, two reagents that restrict intercellular communication across gap junctions. Dye coupling occurred between homologous and heterologous co-cultures of T and B lymphocytes, and was not influenced by their stimulation with PHA and LPS. The connexin mimetic peptide caused a significant decrease in the in vitro synthesis of immunoglobulin M (IgM) by T- and B-lymphocyte co-cultured populations in the presence or absence of stimulation by PHA. The results identify connexins as important cell surface components that modulate immune processes.

The MEK inhibitor, PD98059, reduces survival but does not block acute myeloid leukemia blast maturation in vitro.

The appearance of blasts in acute myeloid leukemia (AML) reflects a shift from cellular processes inducing maturation and cell death to those favouring survival and accumulation. We have monitored changes in the growth factor signalling molecule MAPKinase, in the cytoprotective protein Bcl-2 and in the cell death protein Bax, during maturation of proliferating and non-proliferating AML blasts in vitro. Eighteen AML samples were cultured for 7 d in serum-free medium with or without a supplement of recombinant cytokines comprising c-kit ligand, IL3 and GMCSF. Maturation of AML blasts, as assessed by morphology on Romanowsky-stained slides of 7/18 samples and by changes in surface CD markers on all 18 leukemias, occurred in both the absence and presence of cytokines. Cell numbers decreased to a mean of 71% after 7 d of cytokine-free culture, but increased to 210% in cytokine-supplemented cultures. The proportion of CD15-positive cells, assessed by flow cytometry, increased over 7 d in 17/18 samples, from a mean of 22% to 68% in cytokine-free cultures and to 72% in cytokine-supplemented cultures (p = < 0.0001 for both). By immunofluorescence/flow cytometry, there was no significant change in Bcl-2 over 7 d of culture, while Bax increased, particularly in cytokine-free cultures (2.2-fold), which led to a significant decrease in the Bcl-2/Bax ratio. Immunoblotting demonstrated that ERK was briefly phosphorylated after seeding AML blasts into culture. PD98059, an inhibitor of MAPKinase kinase (MEK) which activates MAPKinase, inhibited this transient ERK phosphorylation but was unable to block maturation as measured by acquisition of CD15 in samples from 12 patients with low starting numbers of CD15-positive cells. PD98059, however, reduced cell numbers in 7-d liquid culture and, in cytokine-supplemented cultures, this was associated with a 1.3-fold increase in Bcl-2 (p = 0.012) and a 1.4-fold increase in Bax (p = 0.02). Overall, these data demonstrate that most leukemic populations can partially differentiate in vitro without the need for cytokines or inducers. The MAPKinase pathway is not required for this maturation, but it does maintain cell viability in the absence or presence of cytokines. A rise in Bcl-2 may not protect AML blasts in the face of elevated Bax.

Antisense-mediated suppression of Bcl-2 highlights its pivotal role in failed apoptosis in B-cell chronic lymphocytic leukaemia.

Although advances have been made in the development of more effective treatment modalities, B-cell chronic lymphocytic leukaemia (B-CLL) remains incurable due to the development of drug resistance. Defective programmed cell death mechanisms rather than dysregulation of cell cycle appears to predominate in B-CLL and it is likely that a failure to initiate apoptosis contributes to chemoresistance. Most B-CLL cells contain high levels of the anti-apoptotic protein Bcl-2 and high Bcl-2/Bax ratios have been associated with in vitro resistance to cytotoxic agents. In this study we evaluated the cellular responses to a Bcl-2 antisense oligonucleotide in terms of Bcl-2 mRNA and protein expression and the induction of apoptosis. The antisense molecule induced a specific reduction in Bcl-2 mRNA and protein expression over the 48 h culture period and was associated with increased apoptosis. The study indicates that Bcl-2 protein is central to the mediation of resistance to apoptosis in B-CLL. Therefore Bcl-2 antisense oligonucleotides might be useful in the treatment of B-CLL.

A novel cell permeant and far red-fluorescing DNA probe, DRAQ5, for blood cell discrimination by flow cytometry.

The deep red fluorescing agent (DRAQ5) is a synthetic anthraquinone with a high affinity for DNA and a high capacity to rapidly enter living cells or stain fixed cells. DRAQ5 is optimally excited by red-light emitting sources and yields a deep red emission spectrum which extends into the low infra-red. DRAQ5 shows excitation at sub-optimal wavelengths including the 488 nm line and the multi-line UV wavelengths emitted by argon-ion lasers. Single beam (488 nm) flow cytometry has been used to demonstrate the utility of DRAQ5-nuclear DNA fluorescence as a discriminating parameter for human leucocytes and lymphoma cells, in combination with fluorochrome-labelled antibodies for the detection of surface antigens and subpopulation recognition. DRAQ5 fluorescence was found to reflect cellular DNA content as evidenced by cell cycle distribution profiles for asynchronous and cell cycle-perturbed populations. Importantly, DRAQ5 can be used in combination with FITC and RPE-labelled antibodies, without the need for fluorescence compensation.

Pleiotropic drug resistance in B-cell chronic lymphocytic leukaemia--the role of Bcl-2 family dysregulation.

B-cell chronic lymphocytic leukaemia (B-CLL) is an incurable clonal disease which shows initial responsiveness to a number of chemotherapeutic drugs. However, most treated patients become resistant to treatment and this represents a major problem in the successful management of the condition. Experimental evidence points to the fact that most chemotherapeutic drugs ultimately exert their cell killing effect through the process of apoptosis. In this study we compared the apoptotic responses of B-CLL cells in vitro following exposure to several chemotherapeutic drugs. We found that there was a correlation between ID50 values for all the drugs under investigation; particularly between Chlorambucil and Fludarabine (P = 0.0002). In addition, we analysed the expression of Bcl-2 and Bax, two proteins pivotal to the regulation of apoptosis, both immediately ex vivo and in viable and apoptotic sub-populations following exposure to drug. Our data suggest that high Bcl-2/Bax ratios may be predictive of a drug resistant phenotype in B-CLL cells and that modulation of these proteins is essential for the induction of cell death. Furthermore, it seems likely that the superior potency that has been ascribed to Fludarabine is due to it being administered in a more optimised dose. A recently reported clinical trial of Fludarabine against high-dose Chlorambucil supports this view since it showed that both treatment modalities were comparable in terms of response rate and survival times.

Chlorambucil resistance in B-cell chronic lymphocytic leukaemia is mediated through failed Bax induction and selection of high Bcl-2-expressing subclones.

Our previous data have shown that high Bct-2/ Bax ratios in chronic lymphocytic leukaemia (B-CLL) correlate with in vitro apoptosis and clinical resistance. We have now monitored the in vitro viability of B-CLL cells in relation to Bcl-2 and Bax expression over a 48 h time course following exposure to chlorambucil. The results showed that Bax up-regulation was essential for chlorambucil-induced apoptosis in B-CLL cells and a 3-fold increase in expression within 4 h of exposure to drug was typically observed in sensitive cells; resistant cells failed to up-regulate Bax at all. In contrast, the constitutively high levels of Bcl-2 found in B-CLL cells were found to be down-regulated in apoptotic cells but the mean Bcl-2 expression in viable cells was increased, probably as a result of the loss of lower Bcl-2-expressing cells into the apoptotic compartment. Taken together, these data add further weight to the suggestion that Bcl-2/Bax ratios may be pivotal in determining the fate of B-CLL cells. Furthermore, the Bcl-2/Bax ratios found in apoptotic B lymphocytes were remarkably similar in the treated, untreated and normal control cells, which suggests that there is a universal Bcl-2/Bax ratio threshold for cell survival and cell death.

Overgrowth of a leukemic culture by a minor CD34+ population.

We have investigated the differentiation potential of blast cells in a case of acute myeloid leukemia which comprised a majority CD34- population and a minor (2%) CD34+ fraction. Blasts were cultured for 2 weeks in a combination of cytokines--c-Kit ligand, interleukin 3 and granulocyte macrophage colony-stimulating factor (SIGm mix)--together with all-trans retinoic acid or 1alpha ,25-dihydroxy vitamin D3. Maturation of blasts was assessed by morphology on Romanowsky-stained slides, changes in surface CD markers and clonogenic culture. After 7 days of culture of unseparated blasts in SIGm, most maturation was monocytic, but with retinoic acid 63% of blasts had matured into granulocytes. Vitamin D3 enhanced monocytic differentiation, with 60% of cells becoming monocytic. The percentage of CD14 and CD15 positive cells decreased over 7 days in SIGm (from 62% to 17% and from 76% to 39% for CD14 and CD15, respectively). CD14+ cell numbers were maintained, or recovered, in cultures supplemented with vitamin D3 (59% at day 7), and CD15+ cell numbers, too, remained unchanged in the presence of retinoic acid (67%) or vitamin D3 (66%). Aberrant markers CD7 and CD56 declined under any conditions. When separated, both the CD34- and CD34+ fractions showed similar changes in morphology and surface maturation markers, suggesting that these two populations may be closely related. However, only a few CD34+ cells expressed the aberrant markers present on the majority blast population. The CD34- population declined in culture while the CD34+ fraction rapidly expanded. This probably reflects the difference in progenitor content; high numbers of colony-forming cells were concentrated in the CD34+ subpopulation. We conclude that both CD34- and CD34+ populations can differentiate but only the CD34+ fraction proliferates. Primitive clonogenic CD34+ cells from this patient may generate occasional aberrant CD34+ blasts which could then differentiate into the accumulating aberrant CD34- blast population.

Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis.

Chlorambucil-induced apoptosis was assessed by three different flow cytometric methods in B-cell chronic lymphocytic leukaemia (B-CLL) cells cultured in vitro and the results were compared with those derived from the morphological assessment of the same samples. Spontaneous apoptosis was consistently observed in the control cultures in the absence of drug but this accounted for less than 12% of all cells in every case. The methods under investigation were the Annexin V labelling assay, the terminal deoxynucleotidyl transferase (TdT) end-labelling assay and the labelling of a 38 kDa mitochondrial membrane protein (7A6 antigen) which is exposed on cells undergoing apoptotic cell death (Apo2.7 assay). The Annexin V assay consistently stained a higher percentage of cells and with a greater separation between the positive and negative cell populations. We conclude that the phosphatidyl serine translocation to the outer leaflet of the cell membrane following an apoptotic signal, as labelled by Annexin V, probably occurs before the development of the DNA strand breaks or the exposure of 7A6 antigen in those cells triggered to die by apoptosis.

GCSF augments post-progenitor proliferation in serum-free cultures of myelodysplastic marrow while ATRA enhances maturation.

All-trans retinoic acid (ATRA) and granulocyte colony stimulating factor (GCSF) are potential inducers of myeloid progenitor cell growth and neutrophil differentiation in myelodysplasia (MDS). We have compared the effects of ATRA and GCSF on the colony growth of 10 MDS marrows, in semi-solid and liquid serum-free mononuclear cell (MNC) cultures, supplemented with a mixture of stem cell factor (SCF), interleukin 3 (IL-3) and granulocyte-monocyte colony stimulating factor (GmCSF) (SIGm mix), which is fully-supportive for myeloid and erythroid (with erythropoietin (EPO)) colony formation in normal marrow. Only 1/10 MDS patients produced normal granulocyte-macrophage colony-forming cell (GmCFC) numbers, under SIGm conditions and erythroid colonies (ECFC) were subnormal in all patients. ATRA (10(-7) M) increased GmCFC numbers (P=0.05) in semi-solid cultures of normal, but not MDS marrow MNC and decreased erythroid colonies in cultures of marrow from either source (P=0.008 and P=0.0001 for normal and MDS, respectively). ATRA enhanced neutrophilic maturation in liquid cultures of both normal and myelodysplastic CD34 + ve cells, as detected by conventional morphology and acquisition of CD15. In contrast to ATRA, GCSF increased Gm colony size but not numbers in semi-solid cultures of normal marrow MNC, which suggests the cytokine augments post-progenitor amplification. This would explain why GCSF increased cell yields in liquid cultures of normal and MDS MNC while GmCFC accumulation remained unchanged. GCSF, though, increased Gm colony numbers in semi-solid cultures of MDS marrow MNC (P=0.014) so that 4/10 patients now grew colonies within the normal range. This was again probably due to increasing clone size, so that some clusters, the numbers of which may be elevated in MDS, were now scored as colonies. Overall, these data indicate that ATRA can enhance the maturation of the progeny of MDS GmCFC whilst GCSF can augment their amplification.

Cardiovascular disease insulin risk in Mexican-American and Anglo-American children and mothers.

OBJECTIVE: To evaluate the relationship between insulin levels and cardiovascular risk factors in children and determine whether it varies among ethnic groups. METHODS: Cardiovascular risk factors and insulin levels were compared in 144 Mexican-American and Anglo-American mother-child pairs, when the children were 11 years of age. RESULTS: Although mean age did not differ between ethnicities, Mexican-American mothers and children both had a greater body mass index (mothers: 29.2 +/- 6.2 vs 27.2 +/- 7.9; children: 21.7 +/- 4.7 vs 19.7 +/- 4.6) and sum of skinfolds than did Anglo-Americans. Triglycerides, very low-density lipoprotein cholesterol, fasting insulin, and cholesterol/high-density lipoprotein ratio were higher, while high-density lipoprotein cholesterol was lower in both Mexican-American adults and children compared with Anglo-Americans. After adjusting for measures of obesity, only high-density lipoprotein cholesterol levels remained significantly lower in Mexican-Americans. For both adults and children, higher quartiles of insulin levels were associated with significantly higher triglycerides, blood pressure and lower high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol/apolipoprotein B levels (estimate of dense low-density lipoprotein size). A summary variable representing cardiovascular risk factors present in adult syndrome X patients was higher in both Mexican-American adults and children than in Anglo-Americans. CONCLUSION: Mexican-American children and adults have higher levels of many cardiovascular risk factors, and this appears related to higher insulin levels and overweight. Appropriate nutrition, weight control, and exercise at early ages could be important in slowing the development of atherosclerosis.