Inhibition of T lymphocyte activation in mice heterozygous for loss of the IMPDH II gene.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, which are also synthesized from guanine by a salvage reaction catalyzed by the X chromosome-linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Since inhibitors of IMPDH are in clinical use as immunosuppressive agents, we have examined the consequences of knocking out the IMPDH type II enzyme by gene targeting in a mouse model. Loss of both alleles of the gene encoding this enzyme results in very early embryonic lethality despite the presence of IMPDH type I and HPRT activities. Lymphocytes from IMPDH II(+/-) heterozygous mice are normal with respect to subpopulation distribution and respond normally to a variety of mitogenic stimuli. However, mice with an IMPDH II(+/-), HPRT(-/o) genotype demonstrate significantly decreased lymphocyte responsiveness to stimulation with anti-CD3 and anti-CD28 antibodies and show a 30% mean reduction in GTP levels in lymphocytes activated by these antibodies. Furthermore, the cytolytic activity of their T cells against allogeneic target cells is significantly impaired. These results demonstrate that a moderate decrease in the ability of murine lymphocytes to synthesize guanine nucleotides during stimulation results in significant impairment in T-cell activation and function.

Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside.

Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C). Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro. In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C. Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene. Sequencing of the dCK coding region showed point mutations in seven patients. Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively. dCK cDNA clones from three patients with > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E. coli and analyzed for dCK activity. Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 99). We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in patients with AML.

Automated platelet counts: accuracy, precision, and range.

The automation of platelet counting is essential in laboratories performing a large number of procedures with high precision. Maintaining this precision and establishing accuracy require an understanding of the special problems of counting platelets, including: (1) the large dynamic range of the measurement, (2) the variable size and aggregability of platelets, and (3) the specific requirements of a quality-control method. In evaluating these problems, we designed experiments that measured the linearity and precision of two types of platelet counters, light scattering and electronic aperture impedance, and that evaluated the suitability of commercially available reference materials. The results show that the instrumentation is excellent and the reference materials are good; such that, given a well-planned quality-control method as presented here, the automated platelet count gives a rapid result with precision and accuracy.

Stability of hematologic parameters in healthy subjects. Intraindividual versus interindividual variation.

An automated complete blood count with white blood cell differential was performed yearly on successive groups of healthy second-year medical students from 1979 through 1987. For three classes (1984-1987), the counts were repeated on the same people nine months later. These data demonstrated that the mean value of all hematologic parameters was quite stable over nine years. This allowed for an estimation of the upper limit for the combined effects of drift in accuracy, precision, and biologic stability. The stability was achievable despite an evolution in technology and quality assurance methods over that period. A comparison of intraindividual versus interindividual variation demonstrated that a normal range based on population statistics may be less sensitive than a normal range established for a person during routine health maintenance, especially for the platelet count.

Bone marrow histiocytic proliferation in association with colony-stimulating factor therapy.

A 6-year-old white male had a myelodysplastic syndrome (refractory anemia with excess blasts in transformation) and was treated with high dose chemotherapy. A combined esterase stain of the marrow blasts showed granulocytic differentiation. Subsequently, persistent pancytopenia with a severely hypocellular bone marrow developed, which was treated with a combination of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Sixteen days after the initiation of therapy, histologic examination of the bone marrow revealed a marked proliferation of histiocytes. To the best of the authors' knowledge, this is the first report of histiocytic proliferation in the bone marrow after colony-stimulating factor therapy.

Bcr/abl recombinant DNA analysis versus karyotype in the diagnosis and therapeutic monitoring of chronic myeloid leukemia.

Karyotype and bcr/abl recombinant DNA analyses are two means of detecting the chromosomal aberration in chronic myeloid leukemia. The authors compared these two methods in a retrospective study of 36 patients with CML in which they found the bcr/abl DNA recombinant event in 100% (29 of 29) of those patients who had the Philadelphia chromosome. To achieve this sensitivity, a battery of two bcr probes and three restriction enzymes is necessary. The authors propose a sequential algorithm for efficient use of these probes and enzymes. In 76% of the patients, bcr/abl rearrangement can be detected with a Bgl II digest and a 3' commercial probe. An additional 21% of patients can be detected by a second assay in which the same membrane is rehybridized to a 3' and 5' combination bcr probe. One patient (3%) required an additional restriction enzyme digest with BamH I to detect the recombinant event by the same 3' probe. Karyotype analysis is used to determine cytogenetic remission in patients with CML under therapy. The authors studied the use of DNA analysis by the Southern blot technique to detect a decrease in the relative number of leukemic cells. By dilution studies and densitometric scanning of autoradiographs, the authors were able to detect a 15% decrease in the relative number of cells having the bcr/abl recombinant event. The authors report the preliminary results of three patients in whom they compared the karyotype and recombinant DNA analysis at multiple time points in their clinical course. In conclusion, the bcr/abl recombinant DNA analysis is superior to karyotype for the diagnosis of CML and can be used for monitoring treated patients.

Leukemic cell maturation: phenotypic variability and oncogene expression in HL60 cells: a review.

Leukemic cells in vitro and in vivo demonstrate an unstable phenotype. We have observed in HL60 cells maintained in culture over long periods of time such phenomena as emergence of drug resistance, oncogene amplification, loss of granulocyte and monocyte lineage markers, and alteration in cell growth parameters. As summarized in this report, a comprehensive review of the literature on HL60 cells shows a wide diversity of phenotype for these cells. We have developed and acquired from other laboratories a series of HL60 sublines with varying phenotypic characteristics. The time in continuous log phase culture for HL60 cells (passage 13 ATCC, Rockville MD) to undergo phenotypic drift from a heavily granulated promyelocytic cell to a more undifferentiated agranular blast form on four occasions varied from 3 to 18 months. The actual loss of promyelocytic phenotype occurred rapidly (within less than 1 month) following a variable period of apparent stable phenotype, The change in morphology was invariably accompanied by decreased sensitivity to ARA-c (5- to 20-fold increase in LD50 and dose necessary to induce NSE positive cells). The c-myc gene is variably amplified in sublines of HL60 cells. The expression of c-myc primarily reflects alterations in cell cycle kinetics and was not found to be correlated with a switch between proliferation and maturation. These results suggest that phenotypic drift may be due to loss of response to regulatory signals that affect the expression of a number of cellular genes.

The clinical utility of discriminant functions for the differential diagnosis of microcytic anemias.

Several groups of authors have derived discriminant functions (DFs) based on red cell indices (primarily MCH, MCV, and RDW) that can be used to differentiate iron deficiency from thalassemia minor. The Technicon H*1 analyzer provides a direct MCHC measurement (termed the CHCM), in addition to the conventional computed value (Hgb/PCV). To evaluate the clinical utility of red cell discriminant analysis, chart review was performed in 176 cases for which hemoglobin characterization and quantitation studies had been requested. Six published discriminants were evaluated for cases of clearly defined iron deficiency anemia and thalassemia minor. Overall diagnostic efficiency ranged from 50%-82%, and the diagnostic performance of three of the discriminants failed to achieve statistical significance. Mean values for both MCHC and CHCM were significantly lower in patients with iron deficiency than in patients with other causes of microcytic anemia. It was also observed that MCHC was significantly greater than CHCM in patients with iron deficiency anemia, but not in patients with other causes of microcytic anemia. Both MCHC and the difference between MCHC and CHCM showed potential value as parameters for the differential diagnosis of iron deficiency from other causes of microcytic anemia. It was noted, however, that in 67% of the cases studied, the use of a DF could not have resolved the diagnosis to the extent that hemoglobin characterization and quantitation studies were no longer indicated.

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide.

The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G1/G0 and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G1 DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G1 to G0.