Expression of human adenosine deaminase in mice transplanted with hemopoietic stem cells infected with amphotropic retroviruses.

Amphotropic recombinant retroviruses were generated carrying sequences encoding human adenosine deaminase (ADA). Transcription of the human ADA gene was under control of a hybrid long terminal repeat in which the enhancer from the Moloney murine leukemia virus was replaced by an enhancer from the F101 host-range mutant of polyoma virus. Hemopoietic stem cells in murine bone marrow were infected with this virus under defined culture conditions. As a result, 59% of day-12 colony forming unit spleen (CFU-S) stem cells became infected without any in vitro selection. Infected CFU-S were shown to express human ADA before transplantation and this expression sustained upon in vivo maturation. Mice transplanted with infected bone marrow exhibited human ADA expression in lymphoid, myeloid, and erythroid cell types. Moreover, human ADA expression persisted in secondary and tertiary transplanted recipients showing that human ADA-expressing cells were derived from pluripotent stem cells. These characteristics of our amphotropic viruses make them promising tools in gene therapy protocols for the treatment of severe combined immunodeficiency caused by ADA deficiency. In this respect it is also relevant that the viral vector that served as backbone for the ADA vector was previously shown to be nonleukemogenic.

Donor-derived cells in the central nervous system of twitcher mice after bone marrow transplantation.

The twitcher mouse is an animal model of galactosylceramidase deficiency, comparable to Krabbe's disease, a lysosomal storage disease in humans. As in most lysosomal storage diseases, neurological deterioration is a prominent feature of the disease in these mice. Transplantation of enzymatically normal congenic bone marrow was earlier found to result in prolonged survival and increased levels of galactosylceramidase in the visceral organs of twitcher mice. It is now reported that bone marrow transplantation results in increased galactosylceramidase levels in the central nervous system (CNS). Concomitantly, the levels of psychosine, a highly toxic lipid that progressively accumulates in the CNS of untreated twitcher mice, stabilized at much lower levels in the CNS of treated twitcher mice. Histologically, a gradual disappearance of globoid cells, the histological hallmark of Krabbe's disease, and the appearance of foamy macrophages capable of metabolizing the storage product were seen in the CNS. By immunohistochemical labeling it was demonstrated that these foamy macrophages were of donor origin. The infiltration of enzymatically competent, donor-derived macrophages was accompanied by extensive remyelination in the CNS. It is concluded that after bone marrow transplantation, donor-derived macrophages infiltrate the affected brain tissue and are capable of inducing a partial reversal of the enzyme deficiency.

Effect of bone marrow transplantation on enzyme levels and clinical course in the neurologically affected twitcher mouse.

The effect of allogeneic bone marrow transplantation (BMT) was investigated in the neurologically affected twitcher mouse, a model for human Krabbe's disease. Twitcher mice have a hereditary deficiency of the lysosomal enzyme galactosylceramidase, which causes growth delay, tremor, and paralysis of the hind legs. Death occurs at 30-40 d of age. After BMT galactosylceramidase activity increased to donor levels in hemopoietic organs. In lung, heart, and liver, galactosylceramidase activity rose to levels intermediate between those of twitcher and normal mice. Increased galactosylceramidase activity in liver parenchymal cells indicated uptake of the donor enzyme by recipient cells of nonhemopoietic origin. Enzyme activity also increased in kidney tissue. BMT resulted in a gradual increase in galactosylceramidase activity in the central nervous system to 15% of normal donor levels. A 5-6-fold increase in galactosylceramidase activity was found in the peripheral nervous system. This increase in enzyme activity was accompanied by a partial alleviation of neurological symptoms. In particular, paralysis of the hind legs was prevented by BMT. BMT led to a modest restoration of growth and prolonged survival. In several cases, the mice survived for more than 100 d, but eventually all animals died with severe neurological disease.

Role of bacterial microflora in development of intestinal lesions from graft-versus-host reaction.

Acute secondary disease was induced in (C57BL X CBA)F1 mice by transplanting CBA bone marrow and spleen cells following lethal whole-body irradiation. The lesions of graft-versus-host (GvH) disease were scored quantitatively by counting of degenerated crypts in subcutaneous fetal gut implants that were free of bacteria. In conventional F1 mice the damage in F1 fetal gut was twice as great as in F1 fetal gut implants carried by decontaminated chimeras. CBA fetal gut implants developed substantial damage when present in conventional chimeras, but not when present in decontaminated chimeras. These results could be explained by assuming the presence of cross-reacting antigens on intestinal bacteria and in the gut epithelial tissue. They also explained the profound protection against delayed GvH mortality provided by removal of the intestinal microflora.

Reconstitution of the W/Wv stem cell differentiation defect by infection with Rauscher leukemia virus.

Hematopoietic stem cells of W/Wv mice failed to produce macroscopically visible hematopoietic spleen colonies in irradiated recipient mice. Infection of W/Wv mice of the spleen focus-forming virus-susceptible genotype Fv-2ss (DBA/2) or Fv-2rs (BD2F1) with Rauscher leukemia virus (RLV) restored the spleen colony-forming capacity of the stem cells. The resulting spleen colonies had normal size and cellularity; the frequency of and ratio between granulocyte-macrophage and erythroid progenitor cells were also normal, without excessive production of erythroid cells. The frequency of spleen colony-forming units (CFU-S) appeared to be strongly reduced in W/Wv mice. The seeding fraction of RLV-infected W/Wv stem cells in the recipient spleens did not differ from that of uninfected or RLV-infected +/+ stem cells. At equivalent numbers of CFU-S, spleen suspensions of RLV-infected W/Wv mice were equally effective as +/+ control suspensions in protecting irradiated mice from death due to bone marrow failure. Thus the number of CFU-S observed appeared to be predictive for the number of W/Wv cells required for effective radioprotection. In irradiated W/Wv mice that received transplants of RLV-infected W/Wv cells, circulating erythrocyte numbers approached those of control mice; the erythrocytes were of normal size, in contrast to the macrocytic red cells of untreated W/Wv mice. The reduced frequency of CFU-S in RLV-infected W/Wv mice can be readily explained by a reduced self-replicating capacity, attributable to the W/Wv genes, which was not reconstituted by infection with RLV. The data indicate a direct involvement of pluripotent stem cells upon infection with RLV.

Induction of lung tumors by radioactive isotopes implanted in the rat lung.

Squamous cell carcinomas were induced in the lungs of male WAG/Rij inbred rats by radiation emitted from the isotopes iridium-192 or iodine-125. These isotopes were implanted by a surgical procedure in the lungs of young rats. Forty rats received implants of 192Ir wires and 20 animals, of 125I seeds. In a 14-month observation period, 30 of the 40 animals with implants of 192Ir wires developed tumors. Malignant hemangioendotheliomas occurred with the highest frequency (50%). From the lungs of 12 rats, squamous cell carcinomas were found. In the observation period of 17 months, 3 rats with implants of 125I seeds developed tumors, among which 1 squamous cell carcinoma could be identified. Tumor fragments were transplanted in syngeneic hosts for propagation of the tumors. Histologic appearances of tumors remained constant in subsequent passages. Responses of transplanted tumors growing in the flanks of syngeneic hosts to doses of radiation, methotrexate, or vinblastine were determined. Although the histologic appearances of the 5 squamous cell carcinomas were similar, tumor-doubling times and responses to irradiation and chemotherapeutic drugs were different. Small cell or large cell carcinomas were not observed.

Nature of the delayed graft-versus-host reactivity of fetal liver cell transplants in mice.

Experiments were designed to determine which actual differences in the cellular composition between fetal liver and bone marrow account for the distinct types of graft-versus-host (GvH) disease. The assay of reactive lymphocytes (by in vitro mitogenic stimulation) in fetal liver transplants in mice, the purification of hemopoietic stem cells (HSC) of the transplants, and the quantitation of HSC numbers in the grafts traced the basis for the distinctly weak type of GvH disease after fetal liver cell grafts. It was found that transplantation of purified HSC concentrates did not modify the severity of GvH mortality. The moderate character of the delayed GvH disease was shown to depend on the presence of an HSC population in fetal liver with different qualities and not on numerical differences between the HSC in fetal liver and bone marrow. Data collected also demonstrated that when GvH disease occurred in the recipients of transplants of fetal liver, it shared the characteristic histologic features of the bone marrow GvH syndrome. The recovery of mitogen responsiveness of spleen cells may have been delayed in fetal liver allotransplantation as compared to syngeneic grafting. By supportive infusion of lymphoid cells, it was suggested that the immunodeficiency coinciding with GvH disease represented a secondary manifestation of the disease rather than a primary impairment in lymphoid differentiation.

Differential behavior of human bronchial carcinoma cells in culture.

A feeder layer culture system suited to grow carcinoma cells derived from solid human lung tumors was developed. This report deals with culturing of the four main histological types of lung carcinomas observed in 37 patients: 19 squamous cell, 6 adenocarcinomas, 7 small cell, and 5 large cell carcinomas. The cultures were initiated from 24 fresh human surgical specimens and from 14 human lung tumors grown as xenografts in nude mice. Three different patterns of behavior in culture were found to be characteristic for squamous cell, adenocarcinomas, and small cell carcinomas, respectively. The culture pattern presented by the primary cultures did not appreciably change after passaging in vitro for periods of up to 2 years, even after infinite cell lines were established. Cultures of large cell carcinoma showed one or more of these patterns. From these patterns cells could be cloned and subsequently cultured as separate stable lines. The system described facilitates the identification of specific types of human lung carcinomas almost immediately (within 1 h) after plating (Phase I) as well as during culture.

In vitro colony formation of transplantable rat leukemias in comparison with human acute myeloid leukemia.

In vitro colony formation in two different soft agar systems was studied with bone marrow from untreated patients suffering from acute myeloid leukemia (AML) and from rats in various stages of two different transplantable myeloid leukemias. In both the thin-agar-layer system, which uses a feeder layer of fetal fibroblasts, and the Robinson culture system, human AML marrow failed to produce colonies. A similar failure was observed with the BN rat leukemia. In contrast, the Shay rat leukemic marrow produced an abnormally large number of colonies in the later stages of the disease. Evidence was obtained that the colonies produced by the Shay leukemic marrow consisted of leukemic cells, and that the disappearance of colonies from human AML and from BN rat leukemic marrow is caused by the numerical disappearance of normal pluripotent hemopoietic stem cells from the marrow and by the inability of the clonogenic leukemic cells to produce colonies in vitro. The results indicate that the BN rat leukemia is a realistic animal model for human AML.

Results of allogeneic bone marrow transplants for leukemia using donors other than HLA-identical siblings.

PURPOSE: To compare outcomes of bone marrow transplants for leukemia from HLA-identical siblings, haploidentical HLA-mismatched relatives, and HLA-matched and mismatched unrelated donors. PATIENTS: A total of 2,055 recipients of allogeneic bone marrow transplants for chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) were entered onto the study. Transplants were performed between 1985 and 1991 and reported to the International Bone Marrow Transplant Registry (IBMTR). Donors were HLA-identical siblings (n = 1,224); haploidentical relatives mismatched for one (n = 238) or two (n = 102) HLA-A, -B, or -DR antigens; or unrelated persons who were HLA-matched (n = 383) or mismatched for one HLA-A, -B, or -DR antigen (n = 108). HLA typing was performed using serologic techniques. RESULTS: Transplant-related mortality was significantly higher after alternative donor transplants than after HLA-identical sibling transplants. Among patients with early leukemia (CML in chronic phase or acute leukemia in first remission), 3-year transplant-related mortality (+/-SE) was 21% +/- 2% after HLA-identical sibling transplants and greater than 50% after all types of alternative donor transplants studied. Among patients with early leukemia, relative risks of treatment failure (inverse of leukemia-free survival), using HLA-identical sibling transplants as the reference group, were 2.43 (P < .0001) with 1-HLA-antigen-mismatched related donors, 3.79 (P < .0001) with 2-HLA-antigen-mismatched related donors, 2.11 (P < .0001) with HLA-matched unrelated donors, and 3.33 (P < .0001) with 1-HLA-antigen-mismatched unrelated donors. For patients with more advanced leukemia, differences in treatment failure were less striking: 1-HLA-antigen-mismatched relatives, 1.22 (P = not significant [NS]); 2-HLA-antigen-mismatched relatives, 1.81 (P < .0001); HLA-matched unrelated donors, 1.39 (P = .002); and 1-HLA-antigen-mismatched unrelated donors, 1.63 (P = .002). CONCLUSION: Although transplants from alternative donors are effective in some patients with leukemia, treatment failure is higher than after HLA-identical sibling transplants. Outcome depends on leukemia state, donor-recipient relationship, and degree of HLA matching. In early leukemia, alternative donor transplants have a more than twofold increased risk of treatment failure compared with HLA-identical sibling transplants. This difference is less in advanced leukemia.

The BN acute myelocytic leukemia (BNML) (a rat model for studying human acute myelocytic leukemia (AML)).

Even if animal models have many properties in common with the human disease, as is the case for the BNML and human AML, they have their limitations with respect to the extrapolation to the clinical situation. This also holds for the BNML; thus, conclusions should only be drawn with great caution. Nevertheless, the studies in the BNML model have added considerably to the understanding of various processes that occur during the development of leukemia, e.g., the interaction of leukemic cells and normal hemopoietic stem cells in relation to the microenvironment. The methodology developed in the BNML model allows the quantification of the relative effectiveness of any given treatment with regard to the antileukemic activity compared with the toxicity for normal host tissues. Furthermore, the cell kinetic studies performed in the BNML as a consequence of timed sequential chemotherapy has been helpful in designing an approach to take advantage of this phenomenon in the treatment of acute leukemia. The comparison of the various treatment modalities, employed for the conditioning prior to bone marrow transplantation, made it possible to determine the relative effectiveness of the various approaches. The fractionation of total body irradiation for conditioning purposes was supposed to have a negligible effect with regard to a reduced antileukemic effect. Detailed studies that were conducted in the BNML model did not confirm this hypothesis indicating that (hyper-)fraction of TBI results in a reduced antileukemic effect. The in vitro purging studies in the BNML aimed at the elimination of residual leukemic cells in autologous bone marrow transplantation contributed to the introduction of this method in clinical practice. However, extended studies in the BNML model also indicated that the contribution of the residual leukemia cell in the patient contributed to a much greater extend to the recurrence of leukemia then did the residual cells in the autologous marrow graft. A major contribution of the BNML was achieved in the study of the area of so-called "minimal residual disease" (MRD). A number of so-far unknown aspects of relapsing leukemia could be identified and studied. A new concept of discriminating locally relapsing leukemia and a delayed occurrence of generalized spreading of leukemia formed the basis for the explanation of the observed heterogeneity in the distribution of leukemic cells during the remission and the subsequent relapse phase. In conclusion, it is obvious that proper comparison of the human disease as well as the counterpart in the animal model requires a detailed knowledge of both.(ABSTRACT TRUNCATED AT 400 WORDS)

Retrovirus-mediated transfer and expression of marker genes in the BN rat acute myelocytic leukemia model for the study of minimal residual disease (MRD).

To study minimal residual disease (MRD) in leukemia, we transferred the Escherichia coli genes encoding beta-galactosidase (lacZ) and neomycin resistance (neo(r)) into the subline LT12 of the Brown Norway rat acute myelocytic leukemia (BNML), employing the retroviral BAG vector. In this way leukemic cells were genetically marked. Ten independent cell lines were characterized during in vitro growth as well as during two subsequent in vivo passages for expression of neo(r) for which the neomycin analogue G418 was used, and for lacZ expression for which the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) was used. Out of 10 lines, four revealed permanent high expression of lacZ in all cells. In four other lines greatly varying lacZ expression between the individual cells from these lines was observed. In the remaining two lines lacZ expression was gradually lost. In contrast, neo(r) expression was gradually lost in eight out of the 10 lines, particularly rapidly during in vivo passaging. In the remaining two lines neo(r) expression was retained. The genetic modification did not alter the in vitro leukemogenicity of the cells. Long term in vivo expression of neo(r) and lacZ was followed in two selected lines up to 12 subsequent passages, i.e. one from the group of homogeneous high lacZ expression and one from the group of heterogeneous lacZ expression. In both lines lacZ expression was retained whereas neo(r) expression was rapidly lost after the third passage. The feasibility of using genetically marked leukemic cells for studies of minimal residual disease (MRD) was explored by injecting rats with leukemic cells, treating them with chemotherapy at full blown leukemia development to reduce the tumor load, mimicking the induction of a state of MRD and studying lacZ expression at relapse. LacZ expression was evident in 100% of the cells whereas neo(r) expression was lost in a considerable fraction. These results indicate that the viral vector BAG can be used to mark leukemia cells genetically although a selection of clones with the desired stability of long-term expression is required.

L4415: further characterization of the rat model for human acute lymphocytic leukemia.

The biological properties of a transplantable lymphocytic leukemia, L4415 in the WAG/Rij rat, are described. The radiation-induced L4415 leukemia is characterized as a relatively slowly growing, non-immunogenic, immature T-cell leukemia which shows a reproducible growth pattern upon intravenous (i.v.) transfer. Survival time following i.v. inoculation is inversely related to the number of leukemic cells in the inoculum, which allows a quantitative estimate in terms of log leukemic cell kill of the effect of treatment. The first signs of leukemic growth are found in the bone marrow, the spleen, and the liver. Leukemic cells can be detected in the peripheral blood 13 days after inoculation. Due to replacement of normal hemopoietic tissue by leukemic cells and their number increasing exponentially thereafter, normal hemopoiesis is inhibited in the later stages of the disease as indicated by severe thrombocytopenia and anemia. Death is caused by a combination of splenic rupture, gastrointestinal and pulmonary hemorrhage, and impaired functions of heavily infiltrated organs. Hepatosplenomegaly and lymphadenopathy are prominent features at autopsy. Cyclophosphamide- and radiosensitivity of the clonogenic leukemic cells have been determined, a 2.9 log cell kill could be induced by single dose cyclophosphamide inoculation and a dosage giving a surviving fraction of 0.37 (D0) of 0.99 Gy with an extrapolation number (N) of 8.5 were calculated. Based on these data, the L4415 rat leukemia may be regarded as a relevant model for human acute lymphocytic leukemia and may thus serve to explore new treatment strategies.

Experimental basis of hematopoietic stem cell transplantation for treatment of autoimmune diseases.

Experiments with animal models of autoimmune disease provided the rational and stimulus for the current, clinical studies of autologous stem cell transplantation for the treatment of a variety of severe, refractory, autoimmune diseases. The discoveries that led to the recognition of the key role of hematopoietic stem cells and the successful treatment of autoimmune diseases with bone marrow transplants are reviewed. The relevance of spontaneous and induced autoimmune disease models for the development of clinical treatment regimens is discussed. Most of the investigations with autologous stem cell transplantation have been performed with induced autoimmune disorders: in rats with adjuvant arthritis and in rats or mice with experimental, allergic encephalomyelitis, the current model for multiple sclerosis. The main aspects of this translational research were the conditioning regimens and the degree of T cell depletion of the graft as determinants of remission induction and the incidence of relapses. The emerging recommendations are compared with the outcome so far of the clinical studies.

Changes in the fibroblastoid colony forming unit population from mouse bone marrow in early stages of Soule virus induced murine leukemia.

Fibroblastoid cells from mouse bone marrow belong to the hemopoietic inductive microenvironment involved in the regulation of hemopoiesis (1). We observed a decline in the incidence of precursors of these cells (fibroblastoid colony forming units) in the early stages of viral leukemogenesis. This was not accompanied by similar changes in bone marrow derived hemopoietic colony forming cell populations (CFU-s and CFU-c). Cultured fibroblastoid colonies from the bone marrow of young AKR mice or Soule murine leukemia virus inoculated BALB/c mice were found to produce type-C ecotropic virus. No such production was observed when similarly cultured granulocyte macrophage colonies (CFU-c) from the bone marrow of these mice were examined. The possibility that the fibroblastoid cell population is a major source of ecotropic leukemia viruses in the early stages of viral leukemogenesis is discussed.

Risk of donor lymphocyte infusions following allogeneic bone marrow transplantation.

Clinical data so far suggest that the resistance to induction of graft-vs.-host disease (GVHD) by donor lymphocyte infusions is less prominent in allogeneic bone marrow transplant patients than indicated by the limited results of experimental studies in animals published many years ago. To confirm this apparent discrepancy, graded numbers of donor type splenocytes were given at various intervals after bone marrow transplantation to mouse radiation chimeras. The results were closely similar to the previously published data, in that the chimeras developed a high degree of tolerance, equivalent to 2-3 log protection. It was also observed that this tolerance can be broken by prior treatment with a lethal dose of cyclophosphamide. It is postulated that the leukemic cells in relapsed patients reduce the suppressor cell population that is responsible for the resistance to re-induction of GVHD.

Heterogeneity within the spleen colony-forming cell population in rat bone marrow.

The pluripotent hemopoietic stem cell (HSC) of the rat can be enumerated in a spleen colony assay (SCA) in rats as well as mice. After injection of rat bone marrow into lethally irradiated mice, macroscopically visible spleen colonies (CFU-S) are found from day 6 through 14, but the number varies on consecutive days. In normal bone marrow a constant ratio of day-8 to day-12 colony numbers is observed. However, this ratio is changed after in vivo treatment of rats with cyclophosphamide, as well as after in vitro treatment of rat bone marrow with cyclophosphamide derivatives. This indicates that the CFU-S that form colonies on day 8 react differently to this treatment than the CFU-S that form colonies on day 12, and suggests heterogeneity among the CFU-S population. Posttreatment regrowth of day-8 and day-12 CFU-S is characterized by differences in population-doubling times (Td = 0.85 days vs 1.65 days). Another argument in support of the postulate of heterogeneity within the rat CFU-S population is derived from the fact that (in contrast to normal rat spleen) the spleen of leukemic rats contains high numbers of CFU-S that show a ratio of day-8 to day-12 CFU-S of 4.5, which is different than that observed for a CFU-S population in normal bone marrow (a ratio of 2.4). It is concluded that, in rat hemopoiesis, two populations of spleen colony-forming cells can be distinguished using the rat-to-mouse SCA. This indicates that mouse and rat hemopoiesis are comparable in this respect and that heterogeneity in the stem cell compartment is a general phenomenon.

Serial bone marrow sampling for long-term follow up of human hematopoiesis in NOD/SCID mice.

The study of long-term human hematopoiesis in immunodeficient mice is greatly facilitated by sequential bone marrow (BM) sampling in individual animals. Until now, however, the only way to obtain these samples was by sacrificing the mice. In this paper we describe a novel technique for obtaining BM cells by aspiration from the femur of living mice. The technique is simple and efficient and does not disable the animals. On average 1.6+/-1x10(6) nucleated cells can be collected from one femur at a time, which is sufficient for flow cytometry analysis, cytospin preparations, and polymerase chain reaction assays. The cellular composition of the samples obtained by puncture is identical to that of BM harvested by flushing the femur after sacrificing the animals. We present the results of 81 punctures of the femur in Hu-NOD/SCID chimeras engrafted with Ficoll-separated or CD34bright purified cells from human umbilical cord blood.

Intrinsic potential of phenotypically defined human hemopoietic stem cells to self-renew in short-term in vitro cultures.

In search for culture conditions that will facilitate hemopoietic stem cell (HSC) replication while preserving their primitive properties, we have made use of a multi-parameter FACS assay to define HSCs on basis of their phenotypic characteristics, i.e., CD34++CD33,38,71(-). Bone marrow and umbilical cord blood samples of CD34(+) cells from 31 donors were loaded with the membrane dye PKH26 and each exposed to various culture conditions for 6 days. The cells that retained the primitive CD34(++)CD33,38,71(-) phenotype were analysed for the number of cell replications they underwent, by measuring loss of PKH26 fluorescence after 6 days. A most striking observation was the large inter-sample variation in the proliferative response of cells that retained the CD34(++)CD33,38,71(-) phenotype. In general, samples could be characterised as either good- or poorly-replicating, according to the proliferation property of their CD34(++)CD33,38,71(-) subset. In comparison to this 'intrinsic' potential, the effects of the applied growth stimuli on CD34(++)CD33,38,71(-) cell replication were negligible. In contrast, the overall recovery of the CD34(++)CD33,38,71(-) cells was clearly dependent on the culture stimuli. Of the various conditions tested, serum-free cultures with pre-established stroma maintained the cells with this primitive phenotype most effectively. In cultures supplemented with various combinations of recombinant HGFs, HSC differentiation prevailed. These findings with phenotypically defined HSCs should assist in the design of systems for expansion and ex vivo gene therapy of early hemopoietic cells.

Cloning, biological characterization and high-level expression of rat interleukin-3 using recombinant adenovirus: description of a new splicing variant.

In the present study, we describe the cloning and sequence analysis of rat IL-3. Two different mRNA isoforms were isolated after transfection of COS cells with the cytokine genomic sequences. One of the isoforms has been predicted before by Cohen et al. (1986), and the other one is identical except that it encodes a protein with an insertion of three amino acids at position 56. As names for the two isoforms, we propose IL-3alpha for the predicted and IL-3beta for the novel molecule. IL-3beta mRNA was detected as the predominant isoform in rat lymphocytes in vivo. High levels of the cytokine were obtained after infection of human cells (A549) with a recombinant adenovirus harboring rIL-3beta cDNA (IG.Ad.CMV.IL-3beta). The biological properties of the IL-3beta protein were tested in a FDC-P1 proliferation assay and in a hematopoietic progenitor colony forming assay. To assess in-vivo bioactivity, lysed 293 cells containing IG.Ad.CMV.rIL-3beta virus were injected subcutaneously into F344 rats. Stimulation of hematopoiesis and leucocytosis were observed during the treatment. After subcutaneous injections of the lysed adeno-producer cells in mice, the only effect observed was a cellular infiltration at the site of injection, confirming the poor cross-reactivity between the two species. The biological properties in vitro and in vivo demonstrate that the cDNA sequences of IL-3beta presented here encode active rat IL-3 protein.