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High-titered spontaneous interferon (IFN) antibodies were detected in a patient with pure red cell aplasia (PRCA), a benign mediastinal tumor, and recurrent cutaneous carcinomas. The circulating IFN antibodies reacted broadly with various human IFN-alpha subtypes (20-140 x 10(3) neutralizing units/ml serum) but not with IFN-beta or IFN-gamma, and they neutralized the antiviral activity of the patient's endogenous IFN-alpha. The IFN-alpha-binding activity was restricted to the IgG1 subclass in a nonmonoclonal manner. Whereas the PRCA repeatedly responded to immunosuppression with high-dose cyclosporin A (CSA) and CSA plus plasmapheresis, IFN antibody production continued during treatment with cyclophosphamide and CSA. Serological analysis revealed past infection with parvovirus B19 and persistent B19 IgM titers. Antibody-mediated impairment of the IFN-alpha system might have favored the development of both PRCA and the various cutaneous carcinomas in this patient. [ABSTRACT FROM AUTHOR]

We evaluated clinical characteristics and outcome on imatinib of 22 patients with myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRB. Median age was 49 years (range 20-80), 91% were male. Fifteen different PDGFRB fusion genes were identified. Eosinophilia was absent in 4/19 (21%) cases and only 11/19 (58%) cases had eosinophils ≥1.5×10 9 /L. On imatinib, 17/17 (100%) patients in chronic phase achieved complete hematologic remission after median 2 months (range 0-13)​. Complete cytogenetic remission and/or complete molecular remission by RT-PCR were achieved in 12/13 (92%) and 12/14 patients (86%) after median 10 (range 3-34) and 19 months (range 7-110), respectively. In patients with blast phase (myeloid, n = 2; lymphoid, n = 3), treatment included combinations of imatinib (n = 5), intensive chemotherapy (n = 3), and/or allogeneic stem cell transplantation (n = 3). All 3 transplanted patients (complex karyotype, n = 2) experienced early relapse. Initially, patients were treated with imatinib 400 mg/day (n = 15) or 100 mg/day (n = 7), the dose was reduced from 400 mg/day to 100 mg/day during follow-up in 9 patients. After a median treatment of 71 months (range 1-135), the 5-year survival rate was 83%; 4/22 (18%) patients died (chronic phase; n = 2; blast phase, n = 2) due to progression (n = 3) or comorbidity while in remission (n = 1). Of note, 3/4 patients had a complex karyotype. In summary, the most important characteristics of myeloid/lymphoid neoplasms with rearrangement of PDGFRB include (a) male predominance, (b) frequent lack of hypereosinophilia,, ((c) presentation in chronic or blast phase, (d) rapid responses and long-term remission on low-dose imatinib, and (e) possible adverse prognostic impact of a complex karyotype.)

Background and Objectives: We sought to identify new fusion genes with involvement of the platelet-derived growth factor receptor beta gene (PDGFRB) in three patients presenting with various subtypes of chronic myeloproliferative disorders associated with chromosomal aberrations involving chromosome bands 5q31-33., Design and Methods: We performed 5 rapid amplification of cDNA ends (5 -RACE)-polymerase chain reaction (PCR) with RNA/cDNA derived from a patient (case #1) with a t(5;12)(q31-33;q24) and a second patient (case #2) with a complex rearrangement involving chromosomes 1, 5 and 11. A newly developed DNA-based long-distance inverse PCR (LDI-PCR) was performed on a third patient (case #3) with a t(4;5;5)(q23;q31;q33)., Results: In cases #1 and #2, we identified mRNA fusions between GIT2 exon 12 and GPIAP1 exon 7, respectively, and PDGFRB exon 11. In case #3, LDI-PCR revealed a fusion between PRKG2 exon 5 and a truncated PDGFRB exon 12. The region encoding the catalytic domain of PDGFRbeta is retained in all three cases, with the partner contributing a coiled-coil domain (GPIAP1, PRKG2) or an ankyrin protein interaction motif (GIT2) that may potentially lead to dimerization and constitutive activation of the fusion proteins. Treatment with imatinib (400 mg/day) has led to sustained complete hematologic remission in all three patients., Interpretation and Conclusions: These data provide further evidence that numerous partner genes fuse to PDGFRB in BCR-ABL negative chronic myeloproliferative disorders. Although these fusion genes occur rarely, their identification is essential in order to detect patients in whom targeted treatment with tyrosine kinase inhibitors is likely to be successful.

Background/aims: The significance of interferon antibodies with respect to response to treatment in patients with chronic hepatitis C treated with interferon-alpha (INF-alpha) remains a matter of debate. The influence of ribavirin on IFN-antibody formation in combination therapy with IFN-alpha has not yet been studied. Therefore we evaluated the relationship between IFN-antibodies and response to ribavirin/IFN-alpha combination therapy and IFN-alpha monotherapy., Methods: We studied 169 patients with chronic hepatitis C who were treated either with IFN alpha 2a (6 MU, thrice weekly) alone or in combination with ribavirin (14 mg/kg per day) for twelve weeks. Thereafter, patients who achieved a virological response (HCV-RNA-negative) were treated with 3 MU IFN-alpha thrice weekly for another 40 weeks. IFN antibodies were analyzed and quantified by a double-antigen sandwich enzyme immunoassay (EIA). In 86 patients two neutralization assays--an antiviral neutralization assay as well as an antiproliferative neutralization assay--were performed in addition. The relationship of the development of IFN-antibodies with the virologically defined response to treatment was analyzed., Results: Ribavirin did neither influence the prevalence nor the level of IFN-antibodies. The frequencies of IFN-antibody formation did not differ in the response groups. However, patients with breakthrough showed significantly higher IFN-antibody titers as compared to responder at end of treatment (median 1,336 BU/ml vs. 148 BU/ml; p = 0.018). Among the breakthrough patients those with IFN-antibodies showed the reappearance of HCV-RNA during therapy significantly earlier (median week 24) than those without IFN-antibodies (median week 32; p = 0.03)., Conclusion: The addition of ribavirin to IFN-alpha does not influence the formation of IFN-antibodies. The development of high-titer IFN-antibodies during IFN-alpha or ribavirin/IFN-alpha therapy of patients with chronic hepatitis C may account for the early occurrence of breakthrough in some patients, while other mechanisms seem to be responsible for this phenomenon in the majority of the afflicted patients.

Although several virus- and host-related predictive factors for the response to interferon alfa (IFN-alpha) have been defined in patients with chronic hepatitis C, no pretreatment parameter can definitely predict the response to antiviral treatment. Assessment of the initial response by quantification of serum hepatitis C virus RNA before and 4 weeks after initiation of therapy may be a clinically applicable and reliable parameter to predict long-term response. Therefore, the aims of the present study were to test the predictive value of a decline in HCV RNA of at least 3 log in the first 4 weeks of treatment (deltaHCV RNA) in patients treated with 3 x 10(6) units of recombinant IFN-alpha2a (rIFN-alpha2a) three times per week subcutaneously and to compare deltaHCV RNA with other established predictive factors, such as HCV genotype and pretreatment viremia. Serum HCV RNA was measured by a validated quantitative reverse transcription-polymerase chain reaction (RT-PCR). Geno/subtyping of HCV was performed by direct sequencing of the nonstructural (NS) 5B region of PCR-amplified isolates and subsequent phylogenetic analysis. Stable HCV RNA levels (deltaHCV RNA < or = 1 log) within the first 4 weeks of IFN-alpha treatment were present in 42 of 70 patients. A decline in HCV RNA levels between 1 to 3 log and more than 3 log was observed in 9 (13%) and 19 patients (27%), respectively. In 21 of 70 patients (30%), HCV RNA was not detectable at the end of 12 months' treatment. Three of 26 patients (11%) with a pretreatment viremia of < or = 10(6) copies/mL (all HCV subtype 3a) and 6 of 44 patients (14%) with a pretreatment viremia of > 10(6) copies/mL (HCV subtypes 1b, 2a, 2c, 3a [two patients], and 4) achieved a virological sustained response to interferon-alpha2a treatment. All patients with a virological sustained response had an initial deltaHCV RNA of more than 3 log. In a stepwise discriminant-function analysis, the initial deltaHCV RNA was confirmed as the strongest predictor of virological sustained response (P < .0001). In conclusion, the data of the present study suggest that IFN-alpha treatment can be terminated after 4 weeks in patients with a decrease in HCV RNA levels of less than 3 log, when apparent HCV eradication is considered the therapeutic target. The predictive value of deltaHCV RNA clearly exceeds the significance of HCV genotype and pretreatment viremia as predictors of successful IFN-alpha treatment.

Background/aims: A causative role of hepatitis C virus infection (HCV) has been discussed in the pathogenesis of mixed cryoglobulinaemia and in B-cell non-Hodgkin's lymphoma. No data are available concerning the newly discovered hepatitis G virus (HGV) and extrahepatic manifestations such as haematological malignancies. But, HCV and HGV most probably belong to the same family of Flavivirus. Consequently, we looked for the prevalence of HCV, HGV and cryoglobulins in patients with B-cell non-Hodgkin's lymphoma., Methods: Serum samples from 69 patients with non-Hodgkin's lymphoma were studied. Diagnosis of non-Hodgkin's lymphoma was established according to the Kiel classification. Active HCV- and HGV infections were investigated using polymerase chain reaction for detection of viral RNA. Cryoglobulins were detected from serum and monoclonal immunoglobulin components were analysed with immunofixation electrophoresis. In addition, we assessed the clinical course of HCV- and HGV-infected patients under chemotherapy., Results: Three of 69 (4.3%) patients with B-cell non-Hodgkin's lymphoma were HCV-infected and nine non-Hodgkin's lymphoma patients (13.0%) were positive for hepatitis G virus RNA. All HGV infected patients were suffering from low-grade non-Hodgkin's lymphoma. No HGV-infected patient was co-infected by HCV and neither HCV- nor HGV-infected patients showed clinical signs of chronic liver disease before, during or after chemotherapy. Serum samples from all patients were devoid of cryoglobulins., Conclusions: HCV seems to have no significance for the pathogenesis of non-Hodgkin's lymphoma in Germany. The increased prevalence of hepatitis G (16.3%) in patients with low-grade non-Hodgkin's lymphoma could suggest a pathological consequence of HGV infection outside of the liver. Evidence of clinically relevant hepatic disease in HGV infected patients was not obtained. Further, chemotherapy does not seem to affect the subsequent clinical course of HGV infection.

Interleukin-2 (IL-2) is a 15 kDa glycoprotein with proven activity as an immune stimulant in the treatment of malignant disorders, congenital and acquired immune deficiencies, infectious disorders, and as an adjuvant to vaccines. Both natural and recombinant type IL-2 preparations have been applied in clinical treatment trials and have turned out to be immunogenic, although to a varying extent. Enzyme immunoassays and western blotting are standard procedures for the detection of IL-2-binding antibodies, whereas the neutralizing capacity of these antibodies is frequently demonstrated by inhibition of IL-2-dependent cell growth in vitro. The rate of treatment-induced IL-2 antibodies has varied from 0% to 100% in reported trials and frequently exceeded 50% in patients exposed to recombinant IL-2, whereas natural type IL-2 appeared to be little immunogenic. Duration of treatment, cumulative IL-2 dose, and route of IL-2 administration are likely to determine both the rate of seroconversion as well as composition and properties of the anti-IL-2 antibodies. Interleukin-2 antibodies are polyclonal in nature and predominantly composed of IgM and IgG types. Frequently they react with both recombinant and natural IL-2 types. As a rule, neutralizing IL-2 antibodies are detected in serum samples with high IL-2-binding titers and are recognized later than their non-neutralizing predecessors. Neutralization in vitro, however, does not predict neutralization in vivo, and there are very rare patients with documented, antibody-mediated loss of response to IL-2 treatment. More frequently, IL-2 antibodies will limit the expression of IL-2-dependent proteins in vivo, but the opposite has also been observed. Although the precise mechanism of antibody induction by IL-2 is unknown, immunogenicity of some drug formulations rather than polyclonal B-cell activation appears to play a critical role. Approaches aiming at limiting the immunogenicity of IL-2 preparations are discussed, and strategies how to recognize and circumvent antibody-mediated IL-2 resistance are presented.

Among 13 patients with pemphigus or bullous pemphigoid, high titers of anti-interferon-alpha (IFN-alpha) antibodies were present in all serum samples of one patient suffering from pemphigus foliaceus. This patient was characterized by a relatively benign course of the disease. The IFN antibodies were of oligoclonal or polyclonal origin, predominantly of the IgG subtype, and displayed a broad spectrum of specificity including various natural and recombinant IFN-alpha subtypes as well as recombinant IFN-omega 1. In vitro, these antibodies neutralized both the antiviral and antiproliferative activities of the respective IFN types. Recognition of the patient's endogenous IFN-alpha demonstrated their autoantibody nature. The IFN antibodies were present at diagnosis and resistant to continued immunosuppressive treatment. Despite clinical remission, the IFN antibodies persisted, suggesting that they were not pathogenically related to the skin manifestations of the pemphigus. There were no sings of immune complex-mediated organ damage. IFN antibodies constitute a new class of autoantibodies that may occur in conjunction with pemphigus and likely interfere with the endogenous IFN system.

An 81-year-old woman with systemic mastocytosis responded to subcutaneous recombinant interferon-gamma (rIFN-gamma) treatment for about 6 months, when intestinal symptoms gradually recurred. A serum sample obtained 3 months later was positive for specific rIFN-gamma-binding antibodies, which had been absent at the initiation of treatment. Cessation of IFN-gamma therapy was followed by a slow decline of IFN antibody titers. The IFN-gamma antibodies were of polyclonal or oligoclonal origin, with a predominance of IgG1 and IgG2 and small amounts of IgA and IgM. They neutralized the antiviral activity of both rIFN-gamma and, less efficiently, natural IFN-gamma in vitro. The time course of the neutralizing titers paralleled the IFN-binding activity of the antibodies. Thus, like other cytokines, rIFN-gamma may be immunogenic in rare patients and elicit the formation of neutralizing antibodies that may impair the therapeutic activity of the drug and interfere with the endogenous IFN-gamma system.

Antibodies to IFN-alpha have been recognized as a novel type of autoantibody developing after allogeneic BMT. Ninety-six patients undergoing BMT for various hematologic disorders were followed for the presence of spontaneous IFN-alpha antibodies until 12 years after transplantation. Seven of them (7.3%) developed IFN-alpha antibodies occurred late after BMT (> or = 15 months), rose to very high titers in some patients, and persisted for years despite combined immunosuppressive treatment. They were oligo- or polyclonal in nature, predominantly IgG with a broad IgG subclass distribution, and neutralized the antiviral and antiproliferative activity of various natural and recombinant IFN-alpha types including the patients' endogenous IFN-alpha in vitro. All antibody-positive recipients suffered from chronic GVHD (n = 5) or chronic viral hepatitis (n = 2), but the only significant association was with prior severe aplastic anemia (3/9, 33%; P = 0.022). There was no discernible HLA association of IFN antibody development. Although the clinical relevance of the IFN-alpha antibodies is uncertain they may interfere with cellular defence mechanisms and immune regulation after BMT.

We report a case of a patient with systemic mastocytosis who was treated with interferon-gamma. Because of severe diarrhoea, nausea and weight loss due to mast cell infiltration of the gastric mucosa the patient received 150 micrograms d-1 interferon-gamma subcutaneously for 10 months. During therapy, the plasma concentrations of IL-3, IL-4 and GM-CSF, which seem to play a role in mast cell growth and differentiation were monitored. The patient had good symptomatic relief and the initially very high eosinophil counts in the peripheral blood showed a partial reduction. However, after 4 months of therapy the patient relapsed. In serum obtained after the relapse, but not in stored serum from the beginning of the therapy, neutralizing antibodies against interferon-gamma were found. Therefore an initial response to the therapy and a secondary failure mediated by treatment-induced antibodies against recombinant interferon-gamma might be suggested. Interferon-gamma may be a well tolerated therapeutic option in systemic mastocytosis. However, treatment-induced neutralizing antibodies against recombinant interferon-gamma should be considered if secondary treatment failure occurs.

In a patient undergoing allogeneic BMT for chronic phase CML, de novo chronic GVHD developed within 80 days after transplantation. Eighteen months post-BMT, high serum levels of neutralizing interferon-alpha (IFN-alpha) antibodies were detected, which persisted despite continuous immunosuppressive treatment. The antibodies were of oligoclonal or polyclonal origin, predominantly of the IgG1 type, and reacted broadly with various human IFN-alpha types, including the patients endogenous IFN-alpha, but failed to recognize natural IFN-beta and recombinant IFN-gamma. Pathogenesis and clinical impact of the IFN-alpha antibodies are unknown. Antibodies of cytokines are a novel class of autoantibodies that may develop after allogeneic BMT and interfere with cytokine homeostasis and immune regulation.

Antibodies to interferon (IFN) may compromise IFN treatment in some patients. In tumor therapy, a critical function of type I IFNs is their antiproliferative effect. For the quantification of neutralizing IFN antibodies we have developed an antiproliferative neutralization assay (APA) based on the reduction of IFN-mediated growth inhibition of Daudi cells by IFN-alpha and IFN-beta antibodies. Proliferation was quantified by [3H]thymidine incorporation, and the neutralizing potency of IFN antibody-positive sera was expressed as the neutralizing titer inhibiting 50% of the antiproliferative activity of 10 IU/ml of IFN (NT50). The APA is easy to perform, reproducible, and more sensitive than a well-established antiviral neutralization assay (AVA). All 30 sera with recombinant IFN-alpha 2a-binding antibodies proved to be neutralizing antibody-positive in the APA whereas seven were scored antibody-negative or uninterpretable in the AVA. The APA is recommended as a second or third line assay for the estimation of the neutralizing potency of spontaneous or treatment-induced IFN-alpha and IFN-beta-specific antibodies.

A filter spot-ELISA was developed for the enumeration of interferon-alpha (IFN-alpha) antibody secretion by murine and human cells. Various human IFN-alpha subtypes were coated onto nitrocellulose membranes by means of broad specificity bovine antibodies. Membranes were blocked with milk proteins, and antibodies released by individual cells during a 3 h culture period were visualized as distinct spots using peroxidase-conjugated, species-specific anti-immunoglobulin antibodies and diaminobenzidine/hydrogen peroxide substrate solution. The spot-ELISA is rapid, easy to perform, and highly sensitive. Possible applications include frequency estimates of IFN-alpha antibody-secreting cells in blood, tissues and hybridoma cultures as well as the evaluation of specificity and immunoglobulin class of the respective antibodies.

Fetal liver cells (FLC) were obtained from beagle fetuses 52 days postconception, and were cryopreserved prior to transplantation into ten sibling recipients that had previously been exposed to total-body irradiation delivered in 3 fractions of 6 Gy each at 4 days, 2 days, and 2 hr before grafting. Donors and hosts were genotypically identical for dog leukocyte antigens (DLA)-A, B, and D. A rapid and lasting engraftment was achieved in all animals following the transfer of 0.2 X 10(8) to 1.6 X 10(8) mononuclear FLC/kg body weight, which were equivalent to 0.9 X 10(4) to 19.8 X 10(4) granulocyte/macrophage progenitor cells (CFU-GM)/kg. Between days 14 and 20 posttransplant pretreatment levels were detected for blood granulocytes, between days 23 and 28 for circulating platelets, and between days 35 and 40 for the erythrocyte count and hemoglobin concentration. Increasing the number of CFU-GM transfused resulted in an accelerated granulocyte and platelet recovery. Bone marrow cells were of donor origin throughout the observation interval, but declining proportions of host lymphocytes circulated in the peripheral blood during the initial recovery phase. In two dogs, skin alterations that might indicate slight graft-versus-host disease (GVHD) were noted following days 20 and 70, respectively. Six recipients had to be sacrificed due to inanition, probably secondary to radiation-induced pancreatic insufficiency two to three months after grafting. The results of this study indicate that cryopreserved FLC are highly effective in restoring hemopoiesis in DLA-compatible sibling dogs. Transplantation of canine FLC may prove valuable in analyzing mechanisms pathogenetically related to graft rejection or to the development of GVHD following the transfer of T-cell-depleted hemopoietic grafts at a preclinical stage.

Pokeweed mitogen (PWM) was shown to induce the generation of plasmacytoid cells (PC) from canine peripheral blood mononuclear cells in vitro. PC were detected by immunofluorescence staining of cytoplasmic immunoglobulin. Optimal culture conditions for PC formation were established and the range of the PC response in normal dogs assessed. Addition to PWM of bacterial lipopolysaccharide enhanced PC formation in most instances. This occurred in the absence of increased cell proliferation and without altering the time course of the response. Mitogen-induced PC generation may provide a useful tool for delineating the capacity of canine blood cells to mount a humoral immune response.

The pattern of development of the granulocyte/macrophage progenitor cell (GM-CFC) pools in the course of canine ontogeny was studied by means of the agar culture technique. Colony formation was stimulated by colony stimulating activity (CSA) in serum from lethally irradiated dogs in combination with erythrocyte-depleted peripheral blood leukocytes from normal adult dogs. The colonies thus obtained in cultures from the different organs were in general large (estimated maximum 50 000 cells) and consisted predominantly of mononucleated macrophages, suggesting that, in these studies, a progenitor cell with high proliferative potential (HPP-CFC) has been monitored. In the yolk sac, a transitory GM-CFC pool became established between day 23 and day 48 of gestation, reaching maximum numbers of approximately 41 X 10(3) per organ on days 36/37. At the same time the GM-CFC concentration in blood collected from the heart also reached a maximum of about 31 X 10(3)/ml, indicating its carrier function for the migration of GM-CFC. In the liver a quasi-exponential increase in the GM-CFC numbers took place between days 36/37 and days 57 to 59 when a total of about 15.2 X 10(6) was found but thereafter and up to day 4 post partum the GM-CFC numbers decreased by almost two orders of magnitude. A continuous increase in the GM-CFC numbers was found in the spleen between day 42 of gestation and day 4 post partum when a maximum of 5.1 X 10(6) to 8.7 X 10(6) was reached. In contrast to the GM-CFC numbers in the liver, the splenic GM-CFC dropped only by 50% of peak values when the dogs reached adulthood. The bone marrow always had the highest incidence of GM-CFC, the concentration per 10(6) cells being 18.7 X 10(3)/10(6) cells on days 45/46, the earliest time point at which cultures could be set up. The absolute GM-CFC numbers in the two femora increased continuously between days 45/46 and day 4 post partum in parallel with the growth of the bones. In the thymus a relatively small population of GM-CFC developed between days 42 and 48 of gestation that was kept quite constant at average numbers between 13 X 10(3) and 30 X 10(3) up to day 4 post partum.

We have studied the pattern of CFU-C disappearance from the peripheral blood of normal and total-body-irradiated dogs given cryopreserved cell suspensions from bone marrow, foetal liver and peripheal blood containing known numbers of CFU-C under an autologous and allogeneic donor-recipient relationship. Only a small fraction of infused donor CFU-C could be detected in the circulation of recipients at the end of the infusion. There was an exponential fall in circulating CFU-C, indicating random loss of infused CFU-C. The CFU-C disappearance pattern in each experimental group was reproducible. The mean half life of autologous blood derived CFU-C in the circulating blood of normal recipients was 8.2 min and the mean blood CFU-C turnover was calculated to be 9.3 X 10(5) CFU-C/kg per day.

Circulating autoantibodies constitute a prominent feature of many autoimmune diseases. Recently, antibodies to interferon (IFN) have been recognized as an additional variant occurring spontaneously in about 10% of patients with autoimmune disorders. The reactivity of these antibodies appears to be restricted to IFN-alpha, with antibodies of individual patients recognizing unique epitope patterns. IFN antibodies are preferentially of the IgG type, and they likely constitute part of the normal B-cell repertoire. Whereas antibody titers differ largely among patients, the affinity of IFN binding has not been determined. Likewise, the mechanisms involved in IFN antibody synthesis as well as the clinical impact of these antibodies in autoimmune disorders remain elusive. Based on recent reports and our own observations, this article reviews current knowledge regarding occurrence and properties of IFN antibodies associated with autoimmune conditions. From clinical and experimental evidence, mechanisms possibly involved in IFN antibody production are derived and the significance of antibody-mediated sequelae is evaluated.

In mammalian ontogeny, the liver constitutes the primary hematopoietic organ for some time. Fetal liver cells (FLC) are rich in hematopoietic stem cells with a high proliferative potential but contain few post-thymic T cells. In animal studies, FLC restored hematopoiesis without severe graft-versus-host disease. However, genetic disparity between donor and host frequently limited durable engraftment and prevented or protracted complete immune reconstitution in most fully allogeneic recipients. Some children with severe combined immunodeficiency have been cured by FLC infusion, whereas favorable effects in aplastic anemia, acute leukemia, and inborn errors of metabolism have been limited and badly understood. Fetal liver transplantation in animals may serve as a model for the analysis and management of complications associated with the transfer of purified hematopoietic stem cell grafts and aid in the development of future therapeutic strategies requiring rapidly proliferating stem cell populations.

The restoration of immune functions was followed in dogs for 101 days after fractionated total body irradiation and autologous transfusion of peripheral blood leukocytes (PBL) or bone marrow (BM) cells. Median numbers of 0.9 X 10(5) granulocyte-macrophage progenitor cells per kilogram of body weight were transferred in either group of recipients. The following parameters recovered more rapidly in PBL recipients as opposed to BM recipients: total blood lymphocyte, T- and B-cell counts, serum levels of immunoglobulins IgM and IgA, in vitro blastogenic responses after stimulation with concanavalin A and pokeweed mitogen, and in vitro plasma cell formation after polyclonal B-cell activation with pokeweed mitogen with or without lipopolysaccharide. No major differences were noted for the restoration of serum IgG levels. Circulating lymphocyte and T-cell numbers remained subnormal for more than three months in both groups, whereas B-cell numbers and serum levels of IgA continued to be depressed in BM recipients only. Thus, autologous PBL restored immune functions more rapidly than did BM. Transplantation of PBL, alone or in addition to autologous BM, might also shorten the period of immunodeficiency after cytoreduction in a variety of malignancies in man.

The restoration of the granulocyte-macrophage progenitor cell (CFU-GM) compartments in blood and bone marrow, and the recovery of blood monocytes were followed for up to one year in ten beagles that had been exposed to fractionated (3 X 6 Gy) total-body irradiation before being transfused with cryopreserved fetal liver cells (FLC) from sibling donors that were genotypically matched for dog leukocyte antigens. Grafts contained 0.2-1.6 X 10(8) mononuclear cells and 0.9-19.8 X 10(4) CFU-GM/kg body weight. Numbers of circulating monocytes rose parallel to granulocyte numbers after day 6 and became normal by day 18 posttransplant. In bone marrow aspirates, low numbers of CFU-GM were detected on day 3 and their incidence per 10(5) mononuclear cells was normal after day 14. Circulating CFU-GM were present in significant numbers by day 7 and their elevated concentration per milliliter of blood after day 14 continued for one year. Dextran sulfate injection mobilized normal numbers of CFU-GM into the blood early after transplantation, and spontaneously circulating CFU-GM in a later phase did not differ from blood progenitors of normal animals with respect to radiation sensitivity and sedimentation velocity. Thus, FLC transplantation effected a rapid restoration of granulopoiesis and monocytopoiesis, which was reflected at both the level of mature blood cells and the compartments of CFU-GM in blood and bone marrow, underlining the high repopulating capacity of fetal liver stem cells.

The kinetics and pattern of hemopoietic recovery after supralethal total-body irradiation (TBI) were compared after transfusion of cryopreserved autografts derived from peripheral blood and bone marrow. Fractionated TBI was given in three doses of 6 Gy each at intervals of 48 h. Grafts of peripheral blood mononuclear cells (MNC) were collected by means of continuous-flow centrifugation and by using the mobilizing agent, dextran sulphate. Autografts were adjusted to contain equal numbers of committed progenitor cells (CFU-GM). Dogs grafted with blood-derived MNC (group A) and with MNC from bone marrow (group B) all received about 1 X 10(5) CFU-GM per kg body weight. In all dogs consistent hemopoietic engraftment was achieved. Comparing the pattern of regeneration of the granulocytes, group A dogs showed a significant regeneratory advantage over group B dogs, particularly during the first 20 days after transplantation. Lymphoid recovery was more rapid in group A until day 14. In both groups, blood lymphocytes remained below normal values beyond day 100. The regeneration patterns of the platelets and reticulocytes revealed no significant differences. These results are in agreement with the hypothesis that there are differences in the relationship between CFU-GM content and hemopoietic potential of autografts from different sources.

Ten beagles were exposed to total body X-irradiation (3 X 6 Gy) and rescued with cryopreserved fetal liver cells from DLA-identical siblings obtained around the 52nd day of gestation. Grafts contained 0.2-1.6 X 10(8) mononuclear cells/kg and 0.9-19.8 X 10(4) granulocyte-macrophage progenitor cells/kg. Hemopoiesis and immune functions were followed for up to one year after fetal liver transplantation (FLT). There was a prompt engraftment in all recipients. Bone marrow metaphases were always of donor origin, whereas some host lymphocytes circulated for 2-3 months. Blood granulocytes and monocytes rose to pre-treatment levels within 2-3 weeks of FLT and platelets and erythrocytes were normal within 3-4 and 5-6 weeks, respectively. The relative incidence of bone marrow CFU-GM was normal by day 14 and the absolute numbers of circulating CFU-GM remained elevated for one year after day 14. Blood lymphocytes reached control numbers between days 35 and 101 with a faster B cell than T cell recovery. Their response to mitogen stimulation was normal by day 75, while the mixed lymphocyte reaction tended to be reduced for one year. Serum levels of IgM (day 35) and IgG (day 49) recovered earlier than IgA levels (day 270). Thus, cryopreserved canine fetal liver cells can restore hemopoiesis and immunocompetence with considerable rapidity in histocompatible, adult siblings pre-treated with total body irradiation, and, since they lack mature T cells, may be used to analyze effector mechanisms that mediate rejection of T cell-depleted allografts under less favourable conditions.