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We describe a 31-year-old patient with pure red cell aplasia of pregnancy, successfully managed with regular blood transfusions. In vitro studies showed specific inhibition of day 14 erythroid colonies (BFU-E) using serum and purified immunoglobulin G (IgG) obtained from the patient at diagnosis (before blood transfusion). The inhibition of BFU-E disappeared when haematological remission occurred 3 weeks after delivery and she remains clinically well with a normal haemoglobin 4 years later.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play a protective role in leishmanial infection. Mice with a null mutation in the gene for the beta common (beta c) chain of the receptors for GM-CSF, interleukin(IL)-3 and IL-5 (beta c-null mice) display normal steady state hemopoiesis and develop lung disease similar to the human condition, alveolar proteinosis, due to a lack of signaling by GM-CSF. We therefore expected to observe a heightened sensitivity to Leishmania major in the beta c-null mice. Surprisingly, the beta c-null mice were more resistant to cutaneous infection than wild-type (wt) mice. Upon intradermal injection of L. major promastigotes, fewer beta c-null mice developed cutaneous lesions than wt mice and these lesions were smaller and healed more rapidly than in wt mice. This resistance to disease was associated with a reduced percentage of in vitro infected beta c-null macrophages. Macrophages from beta c-null mice displayed a more activated phenotype and produced increased amounts of nitric oxide following infection with L. major, both in vivo and in vitro. Paradoxically, however, the parasite burden in the draining lymph nodes was similar in both beta c-null and wt mice, suggesting that at least a subpopulation of cells was susceptible to the parasite. The mechanism preventing normal lesion development remains to be elucidated.

Fifty-two patients with poor prognosis carcinoma of the breast underwent peripheral blood stem cell (PBSC) mobilization using five different regimens. The yields of primitive haemopoietic progenitors were quantified by a recently described pre-colony-forming unit (pre-CFU) assay using limiting dilution analysis (LDA). Results of days 14 and 35 pre-CFU were also correlated with conventional CD34+ cell enumeration, CFU-GM (granulocyte–macrophage) and long-term culture-initiating cell (LTCIC) assays. The yield of pre-CFUs with the combination of granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) was significantly higher than with G-CSF alone, cyclophosphamide (Cyclo) and granulocyte–monocyte colony-stimulating factor (GM-CSF), interleukin (IL)-3 and GM-CSF, or Cyclo alone. No significant correlation between neutrophil engraftment and pre-CFU could be demonstrated. Furthermore, CFU-GM was shown to bear a stronger correlation with pre-CFU and LTCIC than CD34+ cell measurement; thus, CFU-GM remains a useful biological tool for haemopoietic stem cell assay. We conclude that the combination of G-CSF and SCF mobilizes the highest number of pre-CFUs as measured by functional pre-CFU assay, which provides an alternative measurement of primitive haemopoietic progenitors to the LTCIC assay.

Mobilization of haemopoietic precursor cells into the circulation by the combination of cytokines, stem cell factor (SCF) and G-CSF in previously untreated patients with carcinoma of the breast resulted in increased yield of collected peripheral blood precursor cells (PBPC). This mobilization of PBPC by SCF with G-CSF lasted several days after ceasing the cytokines in comparison to the rapid fall of PBPC after ceasing G-CSF. Possible mechanisms for this increased and prolonged mobilization were investigated. Immunological phenotyping with CD38, Thy-1 and MDR-1 of the CD34-positive mobilized PBPC detected no difference in maturity compared to PBPC mobilized by G-CSF alone. However, the down-regulation of c-kit, which is associated with the mechanism of mobilization, was much greater in the PBPC mobilized by SCF and G-CSF. The potential clinical implication of increased and prolonged mobilization is increased yield, allowing transplantation of heavily pretreated patients, transplantation with PBPC from a single apheresis, or PBSC support for multiple courses of high-dose therapy from one mobilization procedure.

During early pregnancy, in response to the implanting embryo, the surrounding uterine stroma undergoes a dramatic transformation into a specialized tissue known as the decidua. The de-cidua encapsulates the developing embryo, facilitating nutrient transfer and limiting tro-phoblast invasion. Here we show that female mice with a null mutation of the interleukin-11 receptor alpha chain are infertile because of defective decidualization. A temporal analysis revealed IL-11 expression is maximal in the normal pregnant uterus at the time of decidualization, and in situ hybridization studies showed expression of the IL-11 and the IL-11 receptor alpha chain in the developing decidual cells. These observations reveal a previously unrecognized critical role for IL-11 signaling in female reproduction.

The interleukin-11 receptor alpha-chain, a member of the hematopoietin receptor superfamily, forms, together with gp130, a functional high-affinity receptor complex for interleukin 11. We, and others, reported the cloning of the murine interleukin 11 receptor alpha-chain cDNA (IL11Ra) and recently described the structure of the IL11Ra locus. We also described the presence of a second IL11Ra-like locus in some mouse strains. In this study we report that the second locus, designated IL11Ra2, encodes an mRNA species. The transcript was 99% identical to the IL11Ra transcript in the coding and 3'-untranslated region, but had a different 5'-untranslated region. The complete genomic organization of the IL11Ra2 locus is presented, and the two loci are shown to be located on a 200-kb NaeI genomic fragment. Comparison of the expression pattern of the IL11Ra and IL11Ra2 genes using an RT-PCR restriction fragment length polymorphism strategy revealed that while the expression of IL11Ra was widespread, expression of IL11Ra2 was restricted to testis, lymph node, and thymus.

The clinical application of gene transfer is hindered by the availability of the multipotential stem cells and the difficulty in obtaining efficient retroviral transduction. To assess potential means by which gene transfer into human hemopoietic stem cells might be enhanced, the retroviral transduction efficiency of human bone marrow cells (BM) or peripheral blood progenitor cells (PBPC) was compared at multiple time points after in vivo administration of granulocyte colony- stimulating factor (G-CSF). This was further compared with the transduction efficiency of cells mobilized with G-CSF plus stem cell factor (SCF) in a cohort of patients randomized to receive either one or two growth factors and with normal BM function. Using the LNL6 retrovirus, retroviral transduction efficiencies of up to 19% were observed for both PBPC and BM (n = 26 patients). There was at least a 100-fold increase in PBPC with G-CSF alone and a further 30-fold increase in the total number of progenitor cells available for retroviral transduction using the combination of SCF plus G-CSF. However, pretreatment of patients with G-CSF with or without SCF did not enhance the retroviral infectability of growth factor-mobilized progenitor cells. The effect of the growth factor, Flk-2/Flt3 ligand (FL), was also examined with respect to retroviral transduction efficiency of human progenitor cells. FL plus IL-3 in vitro increased the retroviral transduction efficiency up to eightfold compared with results observed using other combinations of cytokines tested (P < .001). These findings have clinical implications both for increasing the number of target cells for in vivo gene-marking/gene-therapy studies and improving the efficiency of gene transfer.

We describe five new mutations in the uroporphyrinogen decarboxylase (UROD) gene. All mutations were observed in conjunction with decreased erythrocyte UROD and clinical familial porphyria cutanea tarda (fPCT), (four families) or hepatoerythropoietic porphyria (HEP), (one family). The fPCT mutations included three point mutations that resulted in amino acid substitutions: a lysine to glutamine at amino acid position 253 (exon 7); a glycine to arginine at position 318 (exon 10); an isoleucine to threonine at position 334 (exon 10). The lysine to glutamine at amino acid position 253 was found in conjunction with a single C nucleotide deletion in exon 8 on the same allele of the UROD gene in the same family. This deletion resulted in a shift in the reading frame and the introduction of a premature stop codon 8 amino acids downstream. In the fourth family, a 31-bp deletion (nucleotides 828–858: exon 8) of the coding region, resulted in a frameshift and the introduction of a stop codon 19 amino acids downstream. A point mutation was observed in an individual diagnosed with HEP, resulting in an alanine to glycine change at amino acid position 80 and was present on both alleles. All mutations were confirmed in at least one other family member. The impact of these mutations on the function of the UROD protein was examined using in vitro protein expression and with activity assessed using pentacarboxylic acid porphyrinogen I as a substrate for UROD. Although three mutations reduced UROD activity to < 15% of normal, one resulted in a UROD protein with 50% functional activity and the other had near normal activity. These results indicate that many different genetic lesions of the UROD gene are associated with fPCT.

Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.

The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between - 187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA- 1 rather than the presence of trans-dominant negative regulators.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E- derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

We describe multiple alternative transcripts of uroporphyrinogen decarboxylase mRNA in normal individuals and patients with familial porphyria cutanea tarda. mRNA was reverse-transcribed, subjected to the polymerase chain reaction, and analyzed for nucleotide sequence. Seven different transcripts were characterized, and a cryptic splice acceptor site was identified in intron 1. In all mRNAs the exons abutted at previously defined exon boundaries. Characterization of the splice junctions in the genomic DNA showed that splice donor and acceptor sequences complied with the consensus sequences for these sites except for the splice acceptor sequences of exons 3 and 10. THese deviations were present in two normal individuals and one patient with familial porphyria cutanea tarda and were thus unable to explain the multiple aberrant uroporphyrinogen decarboxylase transcripts. We conclude that apparent deletions observed in transcripts derived from the uroporphyrinogen decarboxylase gene in patients with familial porphyria cutanea tarda should be interpreted with caution.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha-chain of the IL-6 receptor and mRNA was expressed in the 3T3-L1 pre-adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor-dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL-11 (Kd approximately 10 nM). The capacity to bind IL-11 with high affinity (Kd = 300-800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL-11.

SummaryThis report describes five families with symptomatic hereditary protein C deficiency. Using a polymerase chain reaction (PCR)-based method, the entire coding sequence and intron-exon boundaries of the protein C gene was amplified from genomic DNA. In each family a single point mutation in the protein C gene was identified. Two unrelated families were found to share the same mutation, while the other three had different mutations. In the first two families with type I protein C deficiency the normal cytosine residue at nucleotide position 8551 in the protein C gene was replaced by thymidine leading to substitution of the normal proline residue at amino acid position 279 by leucine. In the third family with type I deficiency a previously undescribed mutation was identified. In this family the guanosine residue at position 8559 was replaced by adenosine (glycine 282 substituted by serine). In the fourth family, also with type I deficiency, guanosine 8589 was replaced by adenosine (glycine 292 substituted by serine). The fifth family had type II deficiency and in affected members cytosine 8769 was replaced by thymidine (arginine 352 substituted by tryptophan). All these mutations lead to amino acid substitutions in the serine protease domain of the mature protein. All were able to be confirmed by restriction enzyme analysis of PCR-derived DNA. In addition the novel mutation at nucleotide position 8559 was also demonstrable using single strand conformation polymorphism (SSCP) analysis of PCR-derived DNA. These mutations were likely examples of deamination of methylated cytosine occurring in cytosine-phosphate-guanosine (CpG) dinucleotide sequences. These findings confirm the genetic heterogeneity of hereditary protein C deficiency in these families.

We report here the molecular cloning and chromosomal localization of an additional member of the helix-loop-helix (HLH) family of transcription factors, NSCL. The NSCL gene was identified based on its hybridization to the previously described hemopoietic HLH gene, SCL. Murine NSCL cDNA clones were obtained from a day 11.5 mouse embryo cDNA library. The coding region is 399 base pairs and encodes a predicted protein of 14.8 kDa. The nucleotide sequence shows 71% identity and the amino acid sequence shows 61% identity to murine SCL in the HLH domain. The NSCL protein-coding region terminates six amino acids beyond the second amphipathic helix of the HLH domain. Expression of NSCL was detected in RNA from mouse embryos between 9.5 and 14.5 days postcoitus, with maximum levels of expression at 10.5-12 days. Examination of 12- and 13-day mouse embryos by in situ hybridization revealed expression of NSCL in the developing nervous system. The NSCL gene was mapped to murine chromosome 1. The very restricted pattern of NSCL expression suggests an important role for this HLH protein in neurological development.

The helix-loop-helix (HLH) proteins are a family of transcription factors that include proteins critical to differentiation and development in species ranging from plants to mammals. Five members of this family (MYC, SCL, TAL-2, LYL-1 and E2A) are implicated in oncogenic events in human lymphoid tumors because of their consistent involvement in chromosomal translocations. Although activated in T cell leukemias, expression of SCL and LYL-1 is low or undetectable in normal T cell populations. SCL is expressed in erythroid, megakaryocyte and mast cell populations (the same cell lineages as GATA-1, a zinc-finger transcription factor). In addition, both SCL and GATA-1 undergo coordinate modulation during chemically induced erythroid differentiation of mouse erythroleukemia cells and are down-modulated during myeloid differentiation of human K562 cells, thus implying a role for SCL in erythroid differentiation events. However, in contrast to GATA-1, SCL is expressed in the developing brain. Studies of the function of SCL suggest it is also important in proliferation and self-renewal events in erythroid cells.

Immunoglobulin (Ig) and T-cell receptor (TCR) genes were examined in the lymphoblasts of 70 children with immunophenotypically defined B-cell precursor acute lymphoblastic leukemia (ALL). The most frequent genes to rearrange were Ig heavy (H) chain (93%) and TCR delta (79%), followed by TCR gamma (49%), Ig kappa and/or lambda light (L) chain (46%), TCR alpha (46%), and TCR beta (29%). Thus, despite their putative "B-cell precursor" lineage, these leukemias manifest a remarkably high incidence of TCR gene rearrangements. While certain patterns predominate, there is considerable heterogeneity in Ig and TCR genotypes in this disease. No significant associations were found between Ig and TCR genotype and commonly used prognostic factors including age, sex, race, WBC, French-American-British (FAB) subtype, or cytogenetics. However, the lymphoblasts of three of six patients who failed to achieve initial remission had germline patterns of every Ig and TCR gene, a genotype not observed in the leukemic cells from any of the 64 patients who achieved complete remission (p2 = .0007). This study suggests that particular Ig and TCR genotypes may be of clinical relevance in childhood B-cell precursor ALL. The finding of rearranged TCR genes in a large proportion of cases raises fundamental questions about early lineage commitment and lymphocyte differentiation along B-cell and T-cell pathways.

The SCL gene encodes a basic helix-loop-helix transcription factor which is expressed in early haematopoietic progenitors throughout ontogeny and is essential for the normal development of blood and blood vessels. Transgenic studies have characterised spatially distinct 5′ enhancers which direct lacZ expression to subdomains of the normal SCL expression pattern, but the same elements failed to produce appropriate haematopoietic expression. We now describe an SCL 3′ enhancer with unique properties. It directed lacZ expression in transgenic mice to extra-embryonic mesoderm and subsequently to both endothelial cells and to a subset of blood cells at multiple sites of embryonic haematopoiesis including the yolk sac, para-aortic splanchnopleura and AGM region. The 3′ enhancer also targeted expression to haematopoietic progenitors in both foetal liver and adult bone marrow. Purified lacZ(+)cells were highly enriched for clonogenic myeloid and erythroid progenitors as well as day-12 spleen colony forming units (CFU-S). Within the total gated population from bone marrow, 95% of the myeloid and 90% of the erythroid colony-forming cells were contained in the lacZ(+) fraction, as were 98% of the CFU-S. Activation of the enhancer did not require SCL protein. On the contrary, transgene expression in yolk sacs was markedly increased in an SCL−/− background, suggesting that SCL is subject to negative autoregulation. Alternatively the SCL−/− environment may alter differentiation of extra-embryonic mesoderm and result in an increased number of cells capable of expressing high levels of the transgene. Our data represents the first description of an enhancer that integrates information necessary for expression in developing endothelium and early haematopoietic progenitors at distinct times and sites throughout ontogeny. This enhancer provides a potent tool for the manipulation of haematopoiesis and vasculogenesis in vivo.

Publisher Summary The field of experimental hematology has advanced dramatically since the development of the clonal culture assay in the mid-1960s. This technique proved to be crucial for many reasons. The subsequent application of molecular cloning techniques allowed the genes encoding these extracellular growth factors, or colony-stimulating factors (CSFs), to be defined and recombinant molecules to be produced. The ability to molecularly clone mammalian genes also provided a new perspective for experimental hematologists. The knowledge that transcription factors were important in determining cell fate in other systems was quickly translated to hematopoietic cells, with identification of key transcriptional regulators. The application of molecular genetic techniques has provided entirely new approaches for dissecting the hematopoietic compartment. Some of the important transcription factors are reviewed in this chapter, where new insights into their role in hematopoiesis have been provided by using this approach. The chapter concludes by presenting a hopeful future that genetic approaches will continue to be vital in furthering our initial insights into the role of transcription factors in the regulation of hematopoiesis.

The elevated white cell counts (WCC) and myeloid progenitor cell levels in the blood induced by granulocyte colony-stimulating factor (G-CSF) treatment were studied in three settings: cancer patients previously treated with chemo/radiotherapy (n = 13), untreated cancer patients (n = 5) and normal volunteers (n = 9). The inter-individual variations in progenitor cell mobilization responses to G-CSF and the impact of previous chemo/radiotherapy were investigated. The absolute levels of circulating progenitor cells, but not total white cells, were reduced significantly in the pretreated cancer patients (median 961, range 289-3355 per ml blood) as compared to untreated cancer patients (median 9891, range 2219-16625 per ml blood). In each setting, wide ranges of circulating progenitor cell numbers were observed, and the variation in progenitor cell numbers was considerably greater than observed for the WCC. However, progenitor cell numbers in normal volunteers (942-25296 per ml blood) demonstrated as much variance as observed in pretreated cancer patients. This broad physiological variation in progenitor cell levels induced by G-CSF needs to be considered when designing strategies for allogeneic peripheral blood stem cell transplantation.

This study reports two cases of acute lymphoblastic leukemia of precursor B-cell type. The cases were associated with a t(14;18), but no evidence of a Burkitt's-type translocation was found. There was also no evidence of a preexisting follicular lymphoma. This cytogenetic variety of acute lymphoblastic leukemia has not been reported previously.

We have determined the molecular structure of the gene encoding the murine SCL protein (helix-loop-helix transcription factor). The gene consists of seven exons spanning approx. 20 kb. The intron/exon structure, coding region sequences and sequences present at the splice junctions were highly conserved between mouse and human. The 5' flanking sequence contains CCAAT and TATA consensus motifs with several putative binding sites for SP-1, AP-1 and GATA-1. Multiple mRNA transcripts were generated by alternate exon usage. The transcripts differed primarily in the 5' untranslated region (UTR), but potentially also encode a smaller SCL protein. Despite the high degree of conservation between species, the heptamer/nonamer signal sequences in the 5' region of the human SCL gene (the frequent site of SCL disruption in human leukemia) were poorly represented in the murine sequence. In keeping with this, structural abnormalities of murine SCL were uncommon in murine leukemias that express the SCL transcript.

The SCL gene encodes a member of the helix-loop-helix family of transcription factors that have been implicated in regulation of differentiation and development. Although SCL mRNA is not detectable in normal thymocytes or peripheral T-lymphocytes, transcriptional activation occurs in T-cell tumours. A clue to the normal function of SCL has come from demonstration of high levels of SCL mRNA in erythroid cells. To illuminate the function of SCL in the erythroid lineage, an antisense SCL construct was introduced into the human erythroleukaemia cell line, K562. Cells electroporated with a vector containing antisense SCL grew more slowly than control cells which had received vector alone. Non-specific toxicity was excluded by showing that antisense SCL did not influence growth of Raji cells, a B-cell line that does not express endogenous SCL mRNA. Suppression of K562 growth was accompanied by increased spontaneous erythroid differentiation as measured by benzidine staining. K562 cells containing antisense SCL produced smaller colonies in agar and exhibited reduced clonogenicity compared with control cells. In addition, experiments in which K562 colonies were recloned showed that antisense SCL profoundly suppressed self-renewal of K562 cells. These data provide the first evidence that SCL promotes self-renewal in an erythroid cell line and raise the possibility that SCL may function to regulate proliferation of normal erythroid cells.

The human SCL gene is a member of the family of genes that encode the helix-loop-helix (HLH) class of DNA-binding proteins. A murine SCL cDNA was isolated from a normal macrophage cDNA library by using HLH-specific oligonucleotides as hybridization probes. The coding region is 987 base pairs and encodes a predicted protein of 34 kDa. The nucleotide sequence of the coding region shows 88% identity to the human SCL gene, and the amino acid sequence is 94% identical. The HLH motif and upstream hydrophilic region are entirely conserved in the murine and human proteins. The identity between the mouse and human sequences was less marked in the 5' and 3' untranslated regions. Two murine SCL transcripts that differ in the 3' noncoding region have been detected in fetal liver and various cell lines. Variation was also observed in the 5' untranslated region. Interestingly, immediately downstream of the protein-termination codon, both the human SCL sequence and the murine homolog share an E-box element--the suggested target site for DNA binding of HLH proteins. The murine SCL homolog was mapped to the central part of chromosome 4.

We describe the structural organization of the human SCL gene, a helix-loop-helix family member which we believe plays a fundamental role in hematopoietic differentiation. The SCL locus is composed of eight exons distributed over 16 kb. SCL shows a pattern of expression quite restricted to early hematopoietic tissues, although in malignant states expression of the gene may be somewhat extended into later developmental stages. A detailed analysis of the transcript(s) arising from the SCL locus revealed that (i) the 5' noncoding portion of the SCL transcript, which resides within a CpG island, has a complex pattern of alternative exon utilization as well as two distinct transcription initiation sites; (ii) the 5' portions of the SCL transcript contain features that suggest a possible regulatory role for these segments; (iii) the pattern of utilization of the 5' exons is cell lineage dependent; and (iv) all of the currently studied chromosomal aberrations that affect the SCL locus either structurally or functionally eliminate the normal 5' transcription initiation sites. These data suggest that the SCL gene, and specifically its 5' region, may be a target for regulatory interactions during early hematopoietic development.

We have identified the human gene, SCL. We discovered this gene because of its involvement in a chromosomal translocation associated with the occurrence of a stem cell leukemia manifesting myeloid and lymphoid differentiation capabilities. Here we report the sequence of a cDNA for the normal SCL transcript, as well as for an aberrant fusion transcript produced in the leukemic cells. Although different at their 3' untranslated regions, both cDNAs predict a protein with primary amino acid sequence homology to the previously described amphipathic helix-loop-helix DNA binding and dimerization motif of the Ly1-1, myc, MyoD, immunoglobulin enhancer binding, daughterless, and achaete-scute families of genes. For these cDNAs, at least two different 5' ends are predicted, both of which retain this putative DNA binding domain and predict proteins in the range of 20-30 kDa. SCL mRNA is observed in "early" hematopoietic tissues. Taken together, these studies lead to the speculation that SCL plays a role in differentiation and/or commitment events during hematopoiesis.

Normal adult mice and irradiated, marrow-transplanted mice were injected three times daily for 6 days with 200 ng purified bacterially synthesized recombinant Multi-CSF. Normal mice responded with increased peritoneal macrophages, eosinophils and neutrophils, spleen weight increases and major increases in spleen mast cells and hemopoietic progenitor cells. Irradiated, marrow-transplanted mice failed to develop large increases in peritoneal cell numbers but did show rises in spleen weight, marrow cellularity and spleen and marrow progenitor cell levels. The results confirm data indicating a regulatory role for Multi-CSF in vivo and suggest that Multi-CSF may be of some value in accelerating hemopoietic regeneration following marrow transplantation.

It has been hypothesized that a rearrangement between the delta recombining element (delta Rec) and a pseudo J alpha gene serves to delete the TCR-delta locus before rearrangement of the TCR-alpha genes. We have now sequenced a direct, site-specific rearrangement between the delta Rec element and a pseudo J alpha gene in a human leukemic stem-cell line. Putative "N-sequence" addition was noted at the site of recombination, suggesting that this event occurred at a time when the enzyme(s) involved in N-region addition were active in this cell. This provides support for the view that deletion of the TCR-delta locus is required before rearrangement of the TCR-alpha chain genes.

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.

Three lines of transgenic mice carrying the human T-cell lymphotropic virus type 1 tax gene have previously been reported to develop neurofibromas composed of perineural fibroblasts (S. H. Hinrichs, M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1340-1343, 1987; M. Nerenberg, S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1324-1329, 1987). Tumors from these mice and tumor cell lines derived from them expressed high levels of tax RNA and protein. They also expressed high levels of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene as measured by proliferative responses of FD-CP1 target cells using conditioned media from tumor cells and by Northern (RNA) blot analysis of RNA from tumors and tumor cell lines. Although other tissues, such as salivary glands and muscles, in the transgenic mice also expressed high levels of tax, they did not express the gene for GM-CSF. This indicates that tissue-specific cellular factors, in addition to tax, are required for GM-CSF gene expression. Systemic effects of excessive GM-CSF production were demonstrated by infiltration of polymorphonuclear leukocytes into tumor tissues which are not necrotic, by peripheral granulocytosis, and by splenomegaly resulting from myeloid hyperplasia. The interleukin-2 (IL-2) receptor was also found to be expressed by the tumors and tumor cell lines as measured by IL-2-binding and cross-linking studies. This is the first demonstration that the IL-2 receptor can be activated by tax in a nonlymphoid cell type. These in vivo findings are consistent with other reports which have demonstrated in vitro cis-regulatory elements within the 5'-flanking regions of the genes for GM-CSF and the IL-2 receptor which are responsive to trans activation by the tax gene.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

We report a detailed analysis of a lymphoma-induced erythrophagocytic syndrome mimicking histiocytic medullary reticulosis. Phenotypic analysis of cell surface molecules demonstrated a T cell-like phenotype. However, more extensive analysis showed that this phenotype was not typical of any element of the normal T cell lineage. The markers were consistent with a subset of natural killer cells, the lymphokine-activated killer (LAK) cell. The lymphoma cells, like normal LAK cells, were shown to be capable of non-specific cytotoxicity. Moreover, consistent with the reported regulatory effects of certain non-specific killer cells on hemopoiesis, the lymphoma cells were able to suppress in-vitro hemopoiesis, especially maturation of erythroid precursors, although a soluble factor could not be directly demonstrated. Both of these activities were blocked by a monoclonal antibody (9.IC3) which inhibits NK cell function. These findings imply that this tumour is a neoplastic counterpart of the cell identifiable in vitro as an LAK cell.

We have studied a leukemic stem-cell line, DU.528, that is able to differentiate into myeloid and lymphoid cells. The leukemic cells have a translocation between chromosomes 1 and 14, t(1;14)(p33;q11), which we have molecularly cloned and sequenced. Initial screening used joining (J)-segment probes from the T-cell receptor (TCR) alpha- and delta-chain loci. In apparent concert with the translocation, a deletion has occurred between delta-chain diversity (D)-region genes D delta 1 and D delta 2. D delta 2 was observed on derivative chromosome 1 [der(1)] and D delta 1 on der(14) with a deletion of the intervening 10 kilobases of germ-line DNA. The nature of the D delta 1-D delta 2 deletional event implicates a lymphoid recombinase in the mechanism of the translocation. As a consequence of the translocation, an unusual fusion transcript was generated. Probes from chromosome 1 detected a previously unreported transcript in RNA from both the cell line and the patient. A chromosome 14 probe identified the same transcript, thus confirming a fusion transcript derived from both chromosomes 1 and 14. This translocation may identify a gene for which we propose the name SCL (stem-cell leukemia) that is important for hemopoietic development and oncogenesis and that has been disrupted or altered in this stem-cell line.

A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte-macrophage colony stimulating factor can selectively stimulate mature granulocyte function.

Normal adult C57BL, BALB/c, and C3H/HeJ mice were injected intraperitoneally three times daily for up to 6 days with 102,000 U (200 ng) per injection of purified, bacterially synthesized, Multipotential colony-stimulating factor (CSF) (Interleukin-3) (rMulti- CSF) and compared with control mice injected with serum/saline with or without added endotoxin (1 ng/mL). Mice injected with rMulti-CSF exhibited tenfold rises in blood eosinophil and twofold to threefold rises in neutrophil and monocyte levels. The spleens from mice injected with rMulti-CSF showed a 50% increase in weight, elevated levels of maturing granulocytes, eosinophils, nucleated erythroid cells and megakaryocytes, and up to 100-fold rises in mast cells. Progenitor cell frequencies in the spleen were elevated sixfold to 18-fold. No significant changes were observed in the marrow. Sixfold to 15-fold rises were observed in peritoneal cell populations of mice injected with rMulti-CSF with evidence of increased peritoneal macrophage phagocytic activity. Livers of C57BL mice, but not of the other strains, exhibited increased numbers of infiltrating hematopoietic cells whereas rises in mast cell numbers were observed in the mesenteric lymph node, skin, and gut in BALB/c and C3H/HeJ mice. Endotoxin was excluded as being responsible for the observed changes except possibly those involving peritoneal macrophage phagocytic activity. The results indicate that the injection of normal mice with rMulti-CSF significantly stimulates the same types of hematopoietic populations as are stimulated in vitro by Multi-CSF and indicate that this and other CSFs should be useful in stimulating hematopoietic repopulation and functional activity in vivo.