A single dose (1 mg/kg) of milbemycin D was administered orally to 24 dogs with microfilaremia of Dirofilaria immitis, and the number of circulating microfilariae was counted weekly. The number was decreased by 3 to 8% of the pretreatment levels 1 week after the drug administration. The number remained relatively stable for the first 8 weeks and was gradually increased thereafter without returning to the pretreatment levels by 20 weeks. Three or 4 dogs each were euthanatized on day 1, and 1, 4, 8, 12, 16 and 20 weeks after the drug administration to examine the effects of the drug on intrauterine microfilariae and embryos of the worms. Although no intrauterine microfilariae were destroyed directly by the drug, degeneration and collapse of morular embryos and decrease in the number of intrauterine microfilariae were observed 12 after weeks the drug administration. These findings became more remarkable with time, and no intrauterine microfilariae developed in any worms by 20 weeks. The electron-microscopic findings revealed that the nucleoli of oocytes had a high density in the worms of 1, 4 and 8 week groups. Unequal size of cleavage cells and decrease of polysome number were noticed in the early-stage embryos after 8 weeks. It was assumed that the drug might have some effect on the chromosomes or genes in the germinal stem-cell of the heartworm and interfere with protein syntheses, resulting in inhibition of embryonic development. Twelve dogs were given milbemycin D (1 mg/kg) a total of 4 or 6 times monthly according to a prophylactic program.(ABSTRACT TRUNCATED AT 250 WORDS)

Fanconi anaemia (FA) is a rare genetic disorder that predisposes to the development of aplastic anaemia and neoplasia. The pathophysiologic hallmark of FA is increased susceptibility to chromosomal breakage. Superoxide metabolism has also been shown to be involved in the cellular pathophysiology of FA. Human SOD (rh-SOD), an enzyme which dismutates superoxide, has recently been cloned and expressed in yeast. We treated four FA patients with a 2-week infusion of rh-SOD (25 mg/kg d daily) to determine whether rh-SOD had any effect on haemopoietic progenitor cell growth or on the abnormal cellular phenotype. We found that lymphocyte chromosomal aberrations induced by diepoxybutane were decreased during rh-SOD treatment in two patients and that bone marrow progenitors were increased in one patient.

Several studies indicate that interferon (IFN) treatment, intensive chemotherapy and autologous bone marrow transplantation (ABMT) effectively reduce the Ph-positive clone in Chronic Myelogenic Leukemia (CML). In the present study on patients < or = 55 years, we have combined these three treatment modalities. The aim of the study was to eliminate or minimize the Ph-positive clone to see whether a status of minimal residual or Ph-negative disease could be maintained for a longer period of time. After diagnosis, patients received interferon (IFN-a-2b) and hydroxyurea (HU) to keep the white blood cell (WBC) and platelet count below 2-4 and 100-150 x 10(9)/l, respectively. After six months of treatment, Ph-analysis was performed. Patients with Ph-positive cells in bone marrow then received 1-3 courses of intensive chemotherapy. In patients Ph-negative after two courses, bone marrow was harvested and used for ABMT. After a third course, patients with up to 50% Ph-positive metaphases were accepted for ABMT. As of January 1, 1993, 97 patients were registered in the study. Six months of IFN+HU reduced the percentage of Ph-positive metaphases in 57% of the patients (7% became Ph-negative). The corresponding figures after two intensive cytotherapies were 70% (40% Ph-negative). Eighteen patients were autotransplanted. Seven have relapsed with Ph-positivity 3-22 months after ABMT, while nine are Ph-negative at 1-32+ months after ABMT (two not yet analyzed). Seventeen patients are alive and well, while one died one month after ABMT due to interstitial pneumonia.(ABSTRACT TRUNCATED AT 250 WORDS)

When chronic myeloid leukemia (CML) marrow is set up in long-term culture (LTC), Philadelphia chromosome (Ph)-positive (Ph+) cells typically decline and Ph-negative (Ph-) hematopoietic cells often become detectable. In 1987, we initiated a study to evaluate the feasibility of using 10-day cultured marrow autografts to allow intensive treatment of CML. Patients were selected on the basis of a previous assessment of the frequencies of normal and leukemic LTC-initiating cells (LTC-IC) remaining in their marrow after 10 days of LTC. Of the 87 patients evaluated, 36 (41%) were considered eligible, and 22 (15 in first chronic phase [CP], Group 1; and 7 with more advanced disease, Group 2) were autografted with 10-day cultured marrow after intensive therapy. Satisfactory hematological recovery occurred in 16 patients, and of these, only Ph- cells were detected in 13 (nine in Group 1), with 76-94% Ph- cells in the other three (two in Group 1). Ph+ cells reappeared between 4 and 36 months post-autograft in all but one of the 13 patients in whom complete (morphological and cytogenetic) remission had been achieved; the remaining patient died in remission. Nine of these twelve patients were then treated with alpha-interferon (IFN-alpha) 1-3 x 10(6) units/m2, 3-7 days/week; four returned to complete remission, three developed increasing numbers of Ph+ cells, and two are still too early to evaluate. Fifteen patients (12 in Group 1) remain alive and well, nine in hematological remission (eight in Group 1), 9 to 64 months (median 28) post-autograft.(ABSTRACT TRUNCATED AT 250 WORDS)

Primitive hematopoietic cells released into the peripheral blood (PB) were studied in 50 patients with high-grade non-Hodgkin's lymphoma enrolled in a phase III trial of intensive weekly chemotherapy (VAPEC-B) alone or with granulocyte colony-stimulating factor (G-CSF). Mononuclear cells numbers were monitored and their in vitro growth potential assessed in clonogenic progenitor cell assays and in long-term culture. Total colony-forming cells (granulocyte-macrophage [GM], burst-forming unit, erythroid [BFU-E], Mix-CFC) were increased 40-fold (median) over baseline with chemotherapy alone and 106-fold with chemotherapy and G-CSF after the final dose. CD34+ cells were increased to a median of 4%, equivalent to that in normal bone marrow (BM) controls. Circulating colony-forming cell levels were maximal when the recovering total white blood cell (WBC) count reached 5 to 10 x 10(9)/L. The timing of the maximum was reproducible in individual patients. Therefore the WBC count can be used as a guide to the timing of leukapheresis. PB cells from normal controls' and patients' prechemotherapy were unable to sustain hemopoiesis in two-stage long-term cultures. In contrast, PB cells collected from patients primed with chemotherapy alone or chemotherapy with G-CSF at the time of predicted maximal colony-forming cell release were able to generate and sustain hematopoiesis in long-term cultures at a level comparable or superior to normal BM. These findings indicate that the use of G-CSF after routine outpatient chemotherapy stimulates maximal release of primitive hemopoietic cells into the circulation, including colony-forming cells and long-term culture-initiating cells. Their numbers are comparable with those in normal BM and are such that a single leukapheresis will usually yield enough cells for hemopoietic reconstitution after myeloablative chemotherapy.

Adrenomyeloneuropathy is an X-linked recessive disorder characterized by myelopathy, peripheral neuropathy, and cerebral demyelination, which develop in association with the accumulation of very-long-chain fatty acids. The administration of oleic and erucic acids inhibits the synthesis of very-long-chain fatty acids. Recently such dietary treatment has been widely publicized as a possible cure for this disease.

A phase I-II study was conducted in 15 patients to determine the tolerability, efficacy and optimal dose of s.c. yeast-derived granulocyte-macrophage colony stimulating factor (GM-CSF) for mobilization of peripheral blood stem cells (PBSC). PBSC were measured by the colony forming unit granulocyte-macrophage (CFU-GM) and burst forming unit erythroid (BFU-E) in vitro colony assays and CD34 flow cytometric analysis. GM-CSF was administered for 12 days as a single daily s.c. injection at 125 micrograms/M2 (n = 3), 250 micrograms/M2 (n = 3), 375 micrograms/M2 (n = 6) and 500 micrograms/M2 (n = 3). At these doses, sustained serum GM-CSF levels of approximately 100-400 pg/ml were observed for at least six hours. Most (97.2% or 175/180) of the planned GM-CSF doses were administered with no serious adverse effects at any of the dose levels. Three leukapheresis procedures were performed before and then after 8, 10 and 12 doses of s.c. GM-CSF. Compared with basal leukapheresis collections, s.c. GM-CSF mobilized leukapheresis collections yielded greater numbers of total white blood cells ([WBC] 15/15 patients or 100%, p < 0.001) mononuclear cells ([MNC] 8/15 patients or 53%, p = 0.55), CFU-GM (11/15 patients or 73%, p = 0.037), BFU-E (9/15 patients or 60%, p = 0.13) and CD34+ cells (6/15 patients or 40%, p = 0.15). The in vitro CFU-GM were augmented approximately two to tenfold in GM-CSF mobilized collections with no clear dose effects from 125-500/micrograms/M2. However, in vivo engraftment time to absolute neutrophil count (ANC) 500/mm3 correlated strongly with GM-CSF PBSC mobilizing dose (r2 = 0.99). We conclude that s.c. yeast-derived GM-CSF is a simple, effective and tolerable method for mobilization of PBSC.

Chemotherapy-induced myelosuppression often limits escalation of cancer chemotherapy doses. Cyclophosphamide, an alkylating agent, is an ideal candidate for dose escalation: A log-linear relationship between cell kill and dose has been demonstrated, and the drug spares hematopoietic stem cells. In addition, studies suggest that granulocyte-macrophage colony-stimulating factor (GM-CSF) can enhance the ability to achieve optimal dose intensity as well as ameliorating chemotherapy-induced myelosuppression.

In vitro fertilization (IVF), blastomere biopsy of the 6-8 cell embryo, and single cell DNA diagnosis allows couples at risk of transmitting an X-linked or autosomal disease to start a pregnancy knowing their child will not be affected. We present a quick and reliable nested PCR strategy for sex determination at the single cell level by simultaneous amplification and subsequent restriction fragment analysis of the homologous but non-allelic ZFX and ZFY genes present on the X and Y chromosomes respectively. Amplified ZFX and ZFY sequences are of equal size and produce distinguishable HaeIII digestion products. In a randomized, blinded study of 194 individually isolated lymphoblasts, amniocytes, chorion villus cells, and blastomeres, 191 amplified successfully (98.4% sensitivity). None of the sample blanks showed any PCR product, all 90 of the karyotypically XY cells were correctly genotyped as ZFX/ZFY, all 83 of the 84 XX cells that amplified were correctly genotyped as ZFX only, and analyses of all same-embryo blastomeres were completely concordant (100% specificity). This strategy avoids a source of misdiagnosis observed in methods which detect only Y-specific sequences, where amplification failure in an XY cell results in an erroneous XX diagnosis. This rapid (6 hr) and simple method of analysis, when applied to preimplantation embryo diagnosis, allows the avoidance of offspring affected with an X-linked recessive disorder by transferring only female embryos for implantation and ensuing pregnancy.

We studied the utility of G-CSF for harvesting circulating haematopoietic stem cells in patients with leukaemia or lymphoma based on a comparative study in a single patient. Two successive cycles of leukapheresis following cytotoxic chemotherapy were performed in 22 patients as follows: the first cycle was performed with cytotoxic mobilization in all patients while the second cycle was randomized into two groups: cytotoxic (n = 10) and cytotoxic plus G-CSF (cytotoxic/G-CSF) (n = 12) mobilization. Repetitive cytotoxic mobilization did not alter the yields of mononuclear cells (MNC), myeloid (CFU-GM), and erythroid (BFU-E) progenitors. In contrast, cytotoxic/G-CSF mobilization produced significantly higher yields of MNC (2.6-fold), CFU-GM (5.5-fold), and BFU-E (3.9-fold) than did cytotoxic mobilization alone (P < 0.01). The ratio of CFU-GM to BFU-E was not affected by G-CSF. Furthermore, G-CSF led to an earlier peak of CFU-GM following chemotherapy. G-CSF is thus effective in expanding the pool of circulating haematopoietic progenitors.

Treatment of the central nervous system is crucial to the successful treatment of acute lymphoblastic leukemia in children. The intensity and timing of the therapy are based on the presence or predicted risk of central nervous system leukemia as assessed according to criteria that remain controversial.

Many treatments are used for epidermotropic cutaneous T cell lymphomas (CTCL) such as mycosis fungoides (MF) and Sézary syndrome (SS). All pretend to be effective, but none is really curative. As single drug therapy provides a response rate of about 55% with interferon alpha and about 45% with etretinate, we studied the effectiveness of combining these two drugs for immunomodulatory therapy in epidermotropic CTCL. A review of four reports, including a multicenter study performed in 45 patients, indicates a response rate of 56%, with better results for MF than SS. Side effects are generally moderate when low doses are used. The mechanism of action of this combined therapy on cutaneous lesions remains unclear. In vitro, a synergistic effect of retinoids on interferon alpha antiviral activity has been demonstrated, and in vivo an immunohistochemical study showed that the combined therapy modulates antigens expressed by keratinocytes and increases cytotoxic cells in dermis without modifying the number of Langerhans cells in epidermis.

We report the development of a double-cycle elutriation (DCE) technique separating 3 or greater logs of T cells from a stem-cell-enriched marrow fraction and the results of phase I T-cell depletion studies with HLA-disparate related bone marrow transplantation (BMT) donors in two patient groups. In group 1, 10 patients with refractory hematopoietic malignancies received combination chemotherapy, total body irradiation (TBI), and immunosuppression (pre- and post-BMT), and hematopoietic rescue with a marrow transplant, depleted of T cells by elutriation. Potentially to promote engraftment and a graft-versus-leukemia (GVL) effect, 0.5 to 0.75 x 10(5) T cells/kg were added back. All 10 patients engrafted. Five patients developed acute graft-versus-host disease (GVHD; four grade II, one grade III) and two subsequently developed chronic GVHD. Two patients have relapsed (median follow-up, 206 days; range, 46 to 1,035). Four patients died of BMT-related complications (three of infection, one of veno-occlusive disease [VOD]). Four patient are disease-free survivors (median follow-up, 960 days; range, 670 to 1,035). Group 2 included five infants, four with congenital lymphohematopoietic deficiencies and one with refractory acute lymphocytic leukemia (ALL). In these infants, busulfan and increased cyclophosphamide were substituted for TBI. Only the ALL patient received added T cells. Three patients engrafted: one has stable mixed chimerism, one relapsed with ALL, and one rejected the marrow. One patient had primary autologous recovery, while another failed to engraft. None developed GVHD. We conclude that, in this setting of HLA-disparate BMT with post-BMT antithymocyte globulin (ATG) and corticosteroids, DCE significantly depletes T cells from the marrow and that a defined number of T cells can be added without the occurrence of severe GVHD.

The aim of this study is to determine the safety and efficacy of Transfer Factor (TF) in accelerating the haematopoietic recovery in patients with acute leukaemias (AL), following intensive therapy to induce remission of the disease. Twenty-two patients with different types of AL (16 AML, three BC-CML and three ALL) were studied. The patients were divided in two groups. Group 1 (eight AML, two BC-CML and one ALL) received, after myelosuppression induced by chemotherapy, TF (1 unit daily, subcutaneous) until leucocyte count was > 2.5 x 10(9)/l and platelet count > 80 x 10(9)/l. Group 2 was considered the control group and did not receive TF. Treatment with TF accelerated the recovery of neutrophils, leucocytes, platelets (P < 0.001) and haemoglobin (P < 0.01). As a logical consequence, incidence and severity of infection and haemorrhage were lesser in the TF group than in the control group. There was no evidence that TF accelerated the re-growth of leukaemic cells. It seems that TF is safe in AL, accelerating haematopoietic recovery. However, it should be used with caution until results of additional trials become available.

High-dose cyclophosphamide (HD-CY; 7 g/m2) was administered to patients suffering from high risk multiple myeloma (MM). The safety of this procedure, the recirculation and collection of peripheral blood stem cells (PBSC) and the effect of rhGM-CSF and HD-CY were studied. Group I patients (n = 21) were treated with HD-CY alone. Group II patients (n = 10) received 5 micrograms/kg/day rhGM-CSF iv after HD-CY. Neutropenia was shorter in group II (p = 0.01). In group II, the number of circulating colony forming units (CFU-GM) after 14 days was correlated with the number of circulating CFU-GM after 7 days (r = 0.85, p < 0.0001) and with the number of CD34+ cells (r = 0.839, p = 0.01). The total number of mononuclear cells (MNC) and CFU-GM collected per patient was two and seven-fold higher, respectively, in group II (p = 0.01 and p = 0.03). Recovered MNC and CFU-GM were 1.7 and 7-fold higher, respectively, in group II (p = 0.01 and p = 0.004). Our data show that HD-CY is an efficient means of collecting functional PBSC in MM. We suggest that rhGM-CSF is able to further enhance this yield in MM.

The c-kit ligand or stem cell factor (SCF) and the c-kit ligand receptor (KR) are thought to play pivotal roles in the regulation of human hematopoiesis. When added to interleukin 3 (IL-3) and/or granulocyte-macrophage colony stimulating factor (GM-CSF), SCF has an especially profound effect on the in vitro proliferation of several classes of primitive hematopoietic progenitor cells including the burst forming unit megakaryocyte (BFU-MK), the high proliferative potential colony forming cell (HPP-CFC) and the long-term bone marrow culture-initiating cell (LTBMC-IC). These primitive hematopoietic progenitor cells are present in a CD34+HLA-DR- fraction of marrow which has in vivo marrow populating ability and thereby resembles the pluripotent hematopoietic stem cell. Furthermore, the CD34+HLA-DR- marrow subpopulation which expresses KR contains virtually all of the marrow BFU-MK, HPP-CFC and LTBMC-IC, indicating that the human stem cell is KR positive. The addition of SCF, IL-3 and GM-CSF to suspension cultures initiated with CD34+HLA-DR- cells results in an exponential expansion of the numbers of hematopoietic progenitor cells. Large numbers of such progenitor cells generated ex vivo may be useful as transfusion products for the treatment of chemotherapy induced cytopenias. The therapeutic potential of the in vivo administration of SCF has also been evaluated in a phase I trial of recombinant methionyl SCF. SCF administration led to an increase in both differentiated and primitive hematopoietic progenitor cells within the marrow. Such studies suggest that in vivo SCF administration may be useful for improving the quality of bone marrow grafts to be used either for autologous or allogeneic bone marrow transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

This report describes the response of 18 Diamond-Blackfan anemia (DBA) patients to recombinant human interleukin 3 (rhIL-3). rhIL-3 was administered s.c. once daily on an escalating dose schedule (0.5-10 micrograms/kg/day). The rhIL-3 dose was escalated every 21 days until erythroid response was attained, grade III or IV nonhematologic toxicity was observed, or the maximal rhIL-3 dose was reached. Four patients experienced clinically significant erythroid responses. Two of the responders were steroid-dependent and transfusion-independent, while two were steroid-independent and transfusion-dependent. Baseline clinical or laboratory parameters, in particular in vitro bone marrow erythroid progenitor assays, were not useful in predicting rhIL-3 response. Two of the responding patients remain on maintenance rhIL-3 without diminution of effect at 490 and 855+ days. rhIL-3 was discontinued in the other two responders because of the development of deep venous thrombi.

Recombinant cytokines were used to investigate the pathophysiology of Diamond-Blackfan anemia (DBA) and to treat patients with Fanconi's anemia (FA) and amegakaryocytic thrombocytopenic purpura (AMT). We compared the erythroid burst forming units (BFU-E) colony growth of six DBA patients with four normal controls. BFU-E showed erythropoietin (Epo) dose dependence in all patients, although colony numbers were reduced in comparison with normals. The number and size of BFU-E were increased with the addition to Epo of interleukin 3 (IL-3) or Steel factor (SF), but IL-3 + SF was not synergistic. SF increased the nonadherent cell production in DBA long-term bone marrow cultures, and stromal cells from DBA patients showed normal SF mRNA transcripts. These data suggest that SF is not involved in the pathogenesis of DBA, although it may be useful in treatment. A small group of patients with FA and bone marrow failure were treated with daily s.c. granulocyte-macrophage colony stimulating factor (GM-CSF). Toxicity was minimal, and the majority of the patients responded with significant, sustained increase in neutrophils. Multilineage response was rare. GM-CSF may thus be palliative in patients with FA. Five patients with AMT were treated with IL-3 or IL-3 followed by GM-CSF in a phase I/II study. There was minimal toxicity, and IL-3, but not GM-CSF, resulted in improved platelet counts in two patients and decreased platelet transfusion requirements in the other three. Prolonged IL-3 treatment resulted in platelet increases in two of the latter patients. Thus, IL-3 may contribute to the treatment of patients with AMT.