Studies on the liver to kidney switch of erythropoietin production.

Although the liver is the major site of erythropoietin (Ep) production in the fetus, this function is assumed by kidneys in the adult. The mechanisms underlying the liver to kidney switch of Ep formation are not understood. We studied the natural progression of this transition in sheep by measuring Ep production in response to anemia in normal and bilaterally nephrectomized fetal and newborn sheep beginning at about 80 d gestation (normal gestation: 140 d). Removal of both kidneys before induction of anemia did not affect Ep formation up to about 120 d of gestation. A significant reduction (29%, P < 0.02) in Ep synthesis was first noted at about 130 d of gestation (initiation of switch). This level of nephrectomy-induced reduction of Ep formation persisted until about 15 d after birth. Thereafter, bilateral nephrectomy caused further significant decreases (P < 0.05) in Ep production, gradually resulting in near total absence of Ep production at about day 40 postpartum (completion of switch). Chronic administration of testosterone (12 mg/wk) or estradiole benzoate (1.5 mg/d, 5 d/wk) to the fetus/newborn beginning at 85-90 d of gestation enhanced or suppressed erythropoiesis, respectively, but failed to affect the time at which the liver to kidney switch was initiated and/or completed. By contrast, a significant delay (P < 0.001) in the onset, but not completion of the switch occurred in animals that were either thyroidectomized or rendered chronically anemic beginning in the second third of the gestation period. Administration of thyroxin (1.2 mg/d, 5 d/wk) to thyroidectomized fetus/newborns not only prevented the delay in the initiation of the switch, but also accelerated the rate at which the switch was completed. These results demonstrate that in sheep (a) the liver to kidney switch of Ep production is initiated in utero during the last third of the gestation period, but is completed after birth, (b) this transition occurs gradually; the assumption of Ep producing capacity by the kidney is not preceded by an abrupt loss of hepatic Ep formation; and (c) the switch is not affected by changes in sex hormone levels during the prenatal-postnatal growth periods, but is profoundly influenced by alterations in thyroid hormone and oxygen supply-demand levels.

Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero.

Hemopoietic stem cells from human fetal liver were transplanted in utero into preimmune fetal sheep (48-54 days of gestation). The fate of donor cells was followed using karyotype analysis, by immunofluorescence labeling with anti-CD antibodies, and by fluorescent in situ hybridization using human-specific DNA probes. Engraftment occurred in 13 of 33 recipients. Of five live born sheep that exhibited chimerism, all expressed human cells in the marrow, whereas three expressed them in blood as well. Engraftment was multilineage (erythroid, myeloid, and lymphoid) and human hemopoietic progenitors (multipotent colony-forming units, colony-forming units-granulocyte, macrophage, and erythroid burst-forming units) capable of forming colonies in vitro were detected in all five lambs for greater than 2 yr. These progenitors responded to human-specific growth factors both in vitro and in vivo. Thus the administration of recombinant human IL-3 and granulocyte macrophage-colony-stimulating factor to chimeric sheep resulted in a 2.1-3.4-fold increase in the relative expression of donor (human) cells. These results demonstrate that the permissive environment of the preimmune fetal sheep provides suitable conditions for the engraftment and long-term multilineage expression of human hemopoietic stem cells in a large animal model. In this model, donor human cells appear to retain certain phenotypic and functional characteristics that can be used to manipulate the size of donor cell pool.

Modulation of human hemopoietic progenitor cell growth in vitro by recombinant human beta-interferon.

Interferons are known to have modulatory effects on hemopoiesis. Human bone marrow mononuclear cells were employed to test the effects of human recombinant beta-interferon on myeloid and erythroid hemopoietic stem cell growth. Results demonstrated that 1,000 U/ml of beta-interferon significantly inhibited myeloid growth [colony-forming unit (CFU)-granulocyte macrophage] by 40-50%, whereas a higher concentration (10,000 U/ml) abolished CFU-granulocyte macrophage growth by 80-100%. The inhibitory effects of beta-interferon were partially reversible by increasing the concentrations of colony stimulating activity in the culture and could not be abrogated by addition of toxic oxygen radical scavengers such as superoxide dismutase and catalase. The inhibitory effect of interferon was found to be partially dependent on the presence of accessory cells, since less inhibition was seen using T-cell and monocyte depleted bone marrow cells. Lower concentrations of beta-interferon (10-100 units/ml) were without effect. In contrast to myeloid cells, the human erythroid progenitors (CFU-erythroid, burst-forming unit-erythroid) appear to be more sensitive to the inhibitory effects of beta-interferon. In this regard it was found that 100 U/ml of beta-interferon suppressed erythroid growth by 40-50%. These results suggest that human recombinant beta-interferon is capable of suppressing hemopoietic colony growth.

Phase I/II study of cyclophosphamide, carboplatin, and etoposide and autologous hematopoietic stem-cell transplantation with posttransplant interferon alfa-2b for patients with lymphoma and Hodgkin's disease.

PURPOSE: To evaluate the safety and toxicity of interferon alfa-2b (IFN) following an intensive preparative transplantation regimen in patients with relapsed Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Thirty-two patients with NHL or HD underwent autologous transplantation following cyclophosphamide 7,200 mg/m2, carboplatin 1,600 mg/m2, and etoposide 1,600 mg/m2 (CCV). Fourteen patients received an escalating dose of IFN. IFN was started at 1 x 10(6) U/m2 subcutaneously (SC) three times per week with a monthly dose escalation to a maximum of 3 x 10(6) U/m2 SC three times per week. IFN was continued for a total of 6 months. RESULTS: The preparative regiment was well tolerated. Renal dysfunction was noted more frequently in patients with a history of pretransplant cisplatin treatment, and cardiac dysfunction was responsible for the single transplant-related death (3%). IFN was well tolerated with no serious complications. Transient neutropenia and thrombocytopenia were noted in several patients. The mean maximal-dose IFN achieved was 2 x 10(6) IU/m2. The median duration of treatment with IFN was 5.2 months. The overall probability of survival (OS) and event-free survival (EFS) at 36 months, with a median follow-up duration of 18 months, was 42% OS and 14% EFS in HD and 70% OS and 56% EFS in NHL. The EFS at 36 months was 73% for all NHL patients who received IFN and 50% for patients who refused IFN treatment (P = .12), with OS estimates of 100% in the IFN group and 35% in the untreated group (P = .0002). CONCLUSION: CCV is a safe, effective conditioning regimen in patients with NHL or HD. Posttransplant IFN can be safely administered at 2.0 x 10(6) U/m2 three times per week for 6 months and may have a meaningful antitumor effect.

Differentiation of human leukemic cell lines (HL60, U937) toward macrophages is accompanied by production of colony-stimulating activity (CSA).

Human macrophages have been shown to produce colony-stimulating activity (CSA), a glycoprotein capable of promoting the growth of granulocyte-macrophage colonies (CFU-GM) from target nonadherent human bone marrow cells (BM). Two human leukemic cell lines, HL60 (promyelocytic) and U937 (monocytic), that do not routinely produce CSA were studied. Induction of differentiation toward macrophages was followed by acquisition of new cell-surface antigenic determinants characteristic of normal monocyte-macrophages (Mo); this change was accompanied by CSA production. With two monoclonal antihuman-monocyte antibodies (OKM1 and 63D3) it was shown that unstimulated HL60 cells did not express these antigens, but 56% of the HL60-derived macrophages were positive for OKM1 and 51% for 63D3 antibody. Expression of the OKM1 antigen increased from 25% in the unstimulated U937 cells to 47% in the stimulated U937-derived macrophages. In addition, HL60- and U937-derived macrophages were found to produce CSA. This CSA has properties similar to those of the one produced by normal human Mo, in that it is heat labile and protease sensitive. The colonies produced by HL60 CSA were predominantly (68%) of the macrophage type.

Natural killer cell numbers and activity in mobilized peripheral blood stem cell grafts: conditions for in vitro expansion.

Peripheral blood stem cells (PBSC) are increasingly being used as an alternative to autologous bone marrow (BM) for hematologic rescue after high-dose chemoradiotherapy in the treatment of hematologic and nonhematologic malignancies. Mobilization procedures such as chemotherapy and/or hematopoietic growth factor administration are employed to allow for the graft enrichment in hematopoietic stem cells and progenitors and to accelerate trilineage recovery after transplant. The influence of these mobilization procedures on the lymphoid populations in the graft and on immunologic recovery after transplant remains to be determined. We studied six consecutive patients undergoing PBSC high-volume collections after cyclophosphamide (Cyc) and granulocyte colony-stimulating factor (G-CSF) administration and observed that NK cell numbers (phenotypically defined as CD3-CD56+ by flow cytometry) and activity (evaluated by a 51Cr release assay) fully recovered after 4-5 weeks; high numbers of functionally active NK cells (42.1-212.1 x 10(6)/kg b.w.) were present in the grafts, and their percentage and cytotoxic activity rose from the beginning to the end of the harvesting procedure in most cases. CD3-CD56+ and CD34+ cell numbers peaked at the same time point during harvesting, which differed from one patient to another. T (CD3+) cells were always present during harvest, and CD4 and CD8 numbers showed interdonor variability. When we cultured leukapheresed PBSC in the presence of interleukin-2 (IL-2) (10-1000 U/mL) for 6-8 days, we were able to expand the NK population three- to 5.4-fold; 100 U/mL appears to be the best concentration to generate high numbers of cytotoxic NK cells. Pilot studies also suggest that this short exposure to IL-2 does not affect the CD34+ cells. We conclude that PBSC grafts mobilized by combined Cyc and G-CSF and harvested through high-volume leukapheresis contain high numbers of cytotoxic NK cells that can be expanded in vitro by exposure to IL-2. In the setting of PBSC transplant, ex vivo immunomodulation aimed at increasing the NK cell numbers and activity is feasible and may prove to be useful in inducing a graft-vs.-tumor effect, thereby decreasing the relapse rate after transplant.

Cell-cell interaction in human granulopoiesis: role of T lymphocytes.

T lymphocytes have been implicated in the pathogenesis of granulocytopenias. We studied the effects of unstimulated and pokeweed mitogen (PWM) activated intact (Ti) and partially purified T cell subpopulations (T gamma and Tnon gamma, i.e., T mu plus T0) on in vitro granulocyte-macrophage colony formation (CFUGM) by autologous normal human bone marrow (BM). Coculture of BM and Ti, T gamma, or Tnon gamma caused only a slight decrease in the numbers of colonies and clusters; however, when cultured with a mixture of T gamma and Tnon gamma, the inhibition was significant. In contrast, activation of T cells or T cell subsets with PWM resulted in a marked decrease in colony formation. These results demonstrate that: 1) PWM-activated T cells or its subpopulations will inhibit autologous BM colony formation in vitro. The suppression seen with admixes of unstimulated T gamma and Tnon gamma is presumptive evidence that prior cell-cell interaction(s) may be required for the generation of inhibitory cells in this system; and 2) this model may represent an in vitro counterpart of immune-mediated cytopenias in man.

Altered production of GM-CSF and IL-8 in cytomegalovirus-infected, IL-1-primed umbilical cord endothelial cells.

The human cytomegaloviruses (HCMVs) appear to have the potential to disrupt production of hematopoietic cytokines. We examined the production of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-8 by cultured and CMV-infected human umbilical vein endothelial cells (HUVECs) and compared this production with that of uninfected cells. Endothelial cells are, among other things, an integral component of human bone marrow stroma, and are responsible for production of factors that modulate the proliferation and differentiation of human hematopoietic progenitors. HCMV infection increased the production of GM-CSF in IL-1-primed HUVECs without altering GM-CSF levels in infected but unprimed HUVECs. However, this same virus was capable of causing increased production of the inhibitory cytokine IL-8. Both the viral pellet and the cleared viral supernatant appeared to contribute equally to the increased IL-8 and GM-CSF production, because each of these preparations alone was capable of exerting only half the effect seen with whole virus preparations. That both live virus and soluble protein factors within the viral stock contributed to the enhancement in GM-CSF and IL-8 production was further confirmed by inactivation with either ultraviolet or heat treatment of the viral stocks. Although the identity of the factor within the HCMV stock that contributes to this effect remains unknown, studies conducted in the presence of neutralizing antibodies or polymyxin B ruled out a role for tumor necrosis factor-alpha, IL-6, or endotoxin, all known inducers of GM-CSF. These studies indicate that HCMVs can exert both direct and indirect effects on the production of the hematopoietic factor GM-CSF and the inflammatory/inhibitory cytokine IL-8.

Studies on the pathogenesis of refractory anemia.

Nine patients with refractory anemia were studied using the soft agar marrow culture assay (CFU-c) to identify granulocyte-monocyte progenitor cells. Patients' marrows were then cocultured with normal marrow to identify suppressor cells that inhibit normal colony formation. Three of nine patients had low colony formation and no suppression in coculture. These patients may have a defect intrinsic to the marrow granulocyte-monocyte progenitor cell, termed type I. Three of nine patients had normal colony formation and no suppression in coculture, possibly representing a type II defect in the hemopoietic environment. Three of nine patients had low colony formation in the CFU-c assay and their marrow contained cells that suppressed colony formation by normal marrow in coculture. This defect, termed type III, may result from suppressor cells. Thus, refractory anemia may be a syndrome resulting from at least three different pathogenetic mechanisms involving defects in (1) stem cells, (2) the marrow environment or (3) suppressor cells. This may represent one end of the spectrum of pancytopenia with diminished cellularity (aplastic anemia) or normal cellularity (refractory anemia) resulting from similar mechanisms.

Autoimmune pancytopenia. Lymphocyte inhibition of autologous but not allogenic bone marrow growth in vitro.

A patient with autoimmune renal failure, cavitary lung lesions and arthritis experienced pancytopenia while prednisone therapy was being tapered. Utilizing semisolid culture techniques, a population of nonadherent peripheral blood mononuclear cells was demonstrated, which inhibited autologous but not allogeneic bone marrow erythroid colony-forming units (CFU-E) and myeloid colony-forming units (CFU-c) in vitro. No inhibition of CFU-E or CFU-c colony formation was seen when patient's serum or immunoglobulin G (IgG) was added to cultures. Reinstitution of prednisone therapy resulted in normalization of peripheral blood counts, which was accompanied by the loss of the hemopoietic inhibitor cell activity in the patient's peripheral blood. These results demonstrate the need for testing autologous marrow samples when looking for possible immune-mediated inhibition of hematopoiesis.

Studies on the pathogenesis of aplastic anemia.

Three assays were used to study myelopoiesis in 14 patients with aplastic anemia: (1) the soft agar colony assay for granulocyte-monocyte progenitors (CFU-c); (2) coculture of marrow from patients with normal marrows in the CFU-c assay; and (3) culture of marrow pretreated with antithymocyte globulin (ATG) in the CFU-c assay. Marrow from five patients gave low colony counts when cultured alone and suppressed colony formation by normal marrow cells in coculture. Suppressor cells may have caused the aplasia in these patients. Eight patients had low colony formation and no suppression in coculture. These patients may have absent or defective stem cells. Marrow from one patient produced normal colony formation, did not contain suppressor cells and may have a defective hematopoietic environment. Aplastic anemia thus may result from at least three different defects involving (1) the stem cells, (2) the hematopoietic environment or (3) suppressor cells.

Establishment and characterization of a subclone (U-937-AG) from a permanent human monocyte cell line.

The human cell line U-937 has many features suggestive of the monocyte. It may be stimulated in vitro to develop attributes of an activated macrophage with increased phagocytosis and enhanced Fc receptor expression. We describe the establishment of a subclone designated U-937 AG through the use of a mutagenic agent. The features of this subclone include increased tumoricidal cytotoxicity, increased Fc receptor expression for IgG and enhanced phagocytosis of antibody coated erythrocytes, while retaining the usual light microscopic features. The mutant cell line is also capable of producing an erythroid potentiating factor while temporary production of a granulocyte macrophage colony stimulating factor was noted. The availability of a "resting" parent and a more "activated" subclone of this human monocyte cell line should assist in the study of human monocyte activation.

Pre-treatment of transplant bone marrow cells with hydrocortisone and cyclosporin A alleviates graft-versus-host reaction in a murine allogeneic host-donor combination.

The aim of the study was to alleviate graft-versus-host reaction (GVHR) by pre-treatment of the bone marrow (BM) transplant with hydrocortisone (HC) and cyclosporin A (CsA) in C57BL/6J (donor) --> CBA/J (recipient) mouse combination. BM cells were exposed to HC and CsA for 1 h at 37 degrees C and then injected into lethally irradiated (9.5 Gy) mice at a dose of 2 x 10(6) BM cells/mouse. Haematopoietic recovery was assessed on day 12, and survival was followed for 100 days. Combinations of 1000 microg/ml HC and 100 microg/ml CsA, and 100 microg/ml HC and 10 microg/ml CsA significantly reduced MLR and additively mitigated GVHR in vivo, achieving 40% and 26% survival rates, respectively. However, HC and CsA altered neither the peripheral blood cell counts nor in vitro and in vivo BM cell clonogenic potential. Additional studies have shown that HC and CsA blocked con A-driven differentiation of CD8+ and CD4+ CD8+ lymph node cells (LNC) and progression of LNC to S + G2/M cell cycle phases, and inhibited IL-1, IL-2 and TGF-beta while enhancing GM-CSF gene expression in BM cells. Taken together, these data indicate that the pre-treatment of the BM transplant with HC and CsA results in inactivation of GVHR effector cells and mitigation of GVHR while sparing BM repopulating capacity.

The fetus as an optimal donor and recipient of hemopoietic stem cells.

In the present work we used allogeneic in utero transplantation of fetal stem cells in sheep and monkeys. Thus, both the donor and recipient cells had preimmune status. We showed engraftment of allogeneic stem cells in the tolerant environment of the host. The engrafted cells showed trilineage (lymphoid, erythroid and myeloid) expression of differentiation. Long term maintenance of these engrafted cells was observed. We also demonstrated that ex vivo incubation of donor cells with growth factor can enhance the engraftment. Moreover, we have shown that the engrafted cells respond to phlebotomy in the same manner as endogenous cells. We, therefore, conclude that (a) Preimmune fetuses are highly suitable for stem cell transplantation both as donors and recipients. (b) Engraftment can be modulated by brief maneuvers such as ex vivo manipulation. (c) Functionally, the engrafted cells can respond to hemopoietic stimuli in a similar manner as the endogenous cells. Implications of this transplantation system in clinical medicine is discussed. Everyone who is involved in organ transplantation must, sooner or later, come to grips with immunological barriers which establishes the individuality of each organism by recognizing "self" from "non-self". Transplanters must overcome this barrier, if allogeneic organ transplantation is to be successful. In the case of hemopoietic stem cells (HSC), where immunocompetent cells are transplanted, graft-vs-host disease (GVHD) may also be expected. Tolerance can be expected when the recipient is genetically immunodeficient, thus being unable to mount an immunological barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Human-ovine xenogenic transplantation of stem cells in utero.

The preimmune status of the early gestational age fetus provides a permissive environment that bypasses the immunological barrier and permits the engraftment and expression of hemopoietic stem cells (HSC). We used in utero approach to establish long term (greater than 2 years) engraftment and expression of human fetal liver HSC in sheep. Engraftment occurred in 40% (13 of 33) of the recipients. Of 5 live born sheep, all were chimeric. Engraftment was multilineage, involving lymphoid, myeloid and erythroid donor (human) cells. Interestingly, these progenitors have continued to exhibit responsiveness to human specific growth factors both in vitro and in vivo. Therefore, the integration of human HSC into the hemopoietic framework of the host appeared to be incomplete, with donor progenitors retaining certain phenotypic characteristics that may be exploited to preferentially manipulate the donor (human) cell population in these animals. Donor HSC primarily seeded the host bone marrow. Since the donor cells were of liver origin and the host liver at the time of transplantation was the major hemopoietic organ, this near exclusive seeding to the marrow indicates the greater affinity of marrow for the homing HSC. Nonetheless, no cells of donor origin appeared in the host circulation until the perinatal period, suggesting that donor HSC expand with the developing marrow spaces, but do not undergo terminal differentiation. The absence of a significant immunological barrier and the availability of expanding marrow homing sites render the fetus an excellent host (and donor) for HSC transplantation.

A novel high-dose chemotherapy protocol with autologous hematopoietic rescue in patients with metastatic breast cancer or recurrent non-Hodgkin's lymphoma.

In this phase II trial, we used a double dose-intensive chemotherapy and stem cell rescue protocol to treat breast cancer (BCA) patients or non-Hodgkin's lymphoma patients (NHL). The first cycle consisted of high-dose melphalan followed by ABMT. The second cycle used a novel chemotherapy combination; thiotepa, etoposide, carboplatin and cyclophosphamide (TECC) followed by ABMT. We treated 12 patients in total, nine with BCA, three with NHL. All nine BCA patients were treated with the two cycle protocol. The three NHL patients were treated with the second cycle only. Bone marrow (BM, 1 patient), peripheral blood stem cells (PBSC, 10 patients) or both (1 patient) were reinfused 60-72 h after completion of each cycle of chemotherapy. Recovery was rapid; the ANC rose to greater than 500/microl on day +11 (+8 to + 20) and the platelet count to greater than 20000/microl on day +12 (+6 to +20). The toxicities included the expected neutropenic fevers, severe mucositis, diarrhea, and a low incidence of mild renal insufficiency. No patients developed veno-occlusive disease, hemorrhagic cystitis or overt bleeding. With a mean follow-up of 37 months, 83.3% of the patients are alive. Six patients are in complete remission; one patient with BCA relapsed and expired; one patient with NHL is in CR now over 18 months after relapse and subsequent treatment with interferon; one patient is too early to evaluate. Progression-free survival overall is 75%, which is at least equivalent to many other recent studies using similar regimens. In addition, we have also found that delayed addition of G-CSF during the mobilization of PBSC was feasible and resulted in excellent CD34+ cell counts and engraftments, and reduced treatment costs. These results indicate that this chemotherapy is effective with good remission rates and high progression-free survival rates. It is also well tolerated with acceptable toxicities that are manageable. Long-term follow-up of a larger cohort of patients will be needed to ascertain the overall efficacy of this type of therapy.

Cytomegalovirus as a cause of pancytopenia.

Human cytomegalovirus, HCMV, infects most of the population by adulthood; The primary infection is often accompanied by transient neutropenia and thrombocytopenia, and is followed by a period asymtomatic viral latency. In the setting of bone marrow transplantation, however, the immunosuppressed state of the recipient enables HCMV to re-activate or to infect the individual and cause serious sequelae. These range from hepatitis and gastrointestinal disease to interstitial pneumonia and hematologic abnormalities, which are more common in the allograft. Little is currently known about the mechanisms by which HCMV causes these hematologic abnormalities. In this review, we discuss experimental models which are helping investigators understand the immunology and pathology of CMV infection. We also summarize the vivo studies of the effects of HCMV on human hematopoiesis. Several possible mechanisms that could explain the deleterious effect of HCMV on human hematopoietic function include: 1) alteration of accessory cell function by inducing the production of inhibitory cytokines; 2) perturbation of stromal cell function resulting in a decreased production of hematopoietic factors or by altering cell surface adhesion molecule expression; 3) by direct infection of the hematopoietic stem or progenitor cells. It is likely that the pathogenesis of this syndrome is multifactorial therefore requiring a broad therapeutic approach. This would include the use of the antiviral agents, hematopoietic growth factors and donor derived HCMV specific cytolytic cells.

Human natural killer cells.

Human natural killer (NK) cells comprise 10 to 15% of peripheral blood lymphocytes, characterized by their morphologic appearance of large granular lymphocytes (LGLs) and phenotype CD3- CD56+ CD16+ or CD16-. Functionally, these cells are defined by their ability to lyse target cells without prior sensitization and without restriction by major histocompatibility (MHC) antigens. These cells play an important role in immune defenses, especially after hematopoietic transplantation. They contribute to the defenses against virus-infected cells, graft rejection, and neoplasias; they also participate in the regulation of hematopoiesis through cytokine production and cell to cell interaction. In this mini-review we attempt to summarize the most relevant findings about these cells in terms of their origin and differentiation, their cell surface characteristics including activation and their cytolytic pathways. We have also provided a brief approach of their potential clinical use. Increased knowledge of NK cell differentiation, ontogeny and regulatory mechanisms may be of use for the planning of immunotherapeutic strategies.

Peripheral blood stem cell transfusion for marrow replacement.

A patient is presented who was treated with ablative therapy for Hodgkin's disease and rescued by reinfusion of peripheral blood stem cells (PBSC). The PBSC were used because previous therapy (chemotherapy and radiation to the pelvis) had resulted in fatty hypocellular marrow which was inadequate for marrow transplantation. The PBSC were collected by leukapheresis before and after recovery of the marrow from suppression with cyclophosphamide to bring the stem cells into cohort cycle and to increase the proportion of stem cells in the peripheral blood for collection. The patient showed a successful recovery on a time scale somewhat longer cells administered, the absence of stimulation by granulocyte macrophage-colony stimulating factor or other cytokine, or potential damage done to stromal elements during previous radiation and chemotherapy. The patient remains in clinical complete remission, fully engrafted, more than one year since his autologous transplant.

Erythropoiesis in cancer patients undergoing immunotherapy.

We studied ten patients with various types of cancer who were being treated with Interleukin-2 (IL-2) and lymphokine activated killer cells (LAK). All patients developed a reticulocytopenic, normochromic, normocytic anemia. We noted some variability but no significant suppression of circulating erythroid progenitors. The levels of erythropoietin were lower than expected for the hemoglobin/hematocrit values. We could not detect Interferon or Tumor Necrosis Factor (TNF) in the serum of these patients; however, the supernatant of LAK cells did contain Interferon and TNF which could be neutralized with appropriate antibodies. These results suggest that the etiology of this anemia is multi-factorial. Administration of recombinant erythropoietin (Ep) may be of benefit in some of these patients.