Reconstitution of human hematopoietic function with autologous cryopreserved circulating stem cells

Complete hematopoietic reconstitution using nonleukemic peripheral blood mononuclear cells has been achieved in animal models but not in humans. We treated two patients who had metastatic breast carcinoma involving the bone marrow and who had failed conventional therapy with high-dose chemotherapy and total body radiation. Cryopreserved autologous peripheral blood mononuclear cells (6.3-8.4 X 10(8)/kg patient weight) obtained by leukapheresis before high-dose therapy were returned to the patients intravenously. In one patient, evidence of bone marrow engraftment was present, but the patient died before full reconstitution of the peripheral blood cells occurred. Bone marrow engraftment and return of all cell lines to the peripheral blood occurred in the second patient. These findings demonstrate that human hematopoietic reconstitution can be achieved with autologous, peripheral blood, mononuclear cell transfusions following high-dose therapy. This approach may be useful to patients who have contraindications for a bone marrow harvest but who are otherwise candidates for autologous bone marrow transplantation.     

Severe Aplastic Anemia Associated With Eosinophilic Fasciitis: Report of 4 Cases and Review of the Literature

Diffuse eosinophilic fasciitis (Shulman disease) is a rare sclerodermiform syndrome that, in most cases, resolves spontaneously or after corticosteroid therapy. It has been associated with hematologic disorders, such as aplastic anemia. The clinical features and long-term outcomes of patients with eosinophilic fasciitis and associated aplastic anemia have been poorly described. We report the cases of 4 patients with eosinophilic fasciitis and associated severe aplastic anemia. For 3 of these patients, aplastic anemia was refractory to conventional immunosuppressive therapy with antithymocyte globulin and cyclosporine. One of the patients received rituximab as a second-line therapy with significant efficacy for both the skin and hematologic symptoms. To our knowledge, this report is the first to describe rituximab used to treat eosinophilic fasciitis with associated aplastic anemia.In a literature review, we identified 19 additional cases of eosinophilic fasciitis and aplastic anemia. Compared to patients with isolated eosinophilic fasciitis, patients with eosinophilic fasciitis and associated aplastic anemia were more likely to be men (70%) and older (mean age, 56 yr; range, 18–71 yr). Corticosteroid-containing regimens improved skin symptoms in 5 (42%) of 12 cases but were ineffective in the treatment of associated aplastic anemia in all but 1 case. Aplastic anemia was profound in 13 cases (57%) and was the cause of death in 8 cases (35%). Only 5 patients (22%) achieved long-term remission (allogeneic hematopoietic stem cell transplantation: n = 2; cyclosporine-containing regimen: n = 2; high-dose corticosteroid-based regimen: n = 1).     

Eosinophilic fasciitis

Two patients with clinical and pathologic features of eosinophilic fasciitis manifested serologic and systemic abnormalities that raised the question of the fundamental nature and relationship of eosinophilic fasciitis to scleroderma. In addition to the characteristic features of eosinophilic fasciitis, both patients exhibited arthritis, a predominantly mononuclear cell infiltration of muscles with normal serum muscle enzyme levels, weakly positive serum antinuclear factor, IgA deficiency, and abnormalities of pulmonary function. In addition, one patient had wide-mouthed colonic diverticulae and synovial deposits consistent with amyloid; the second patient had bone marrow hypoplasia. Although corticosteroid therapy was of benefit, hydroxychloroquine and potassium para-aminobenzoate were of further help in controlling the disorder. Biopsies from the two patients revealed inflammatory lesions to be heaviest deep in the skeletal muscle; fascia was only minimally inflamed with mild fibrosis. The findings suggest that striking fibroinflammatory lesions noted in the fascia in some patients with eosinophilic fasciitis may derive largely from spillover of lesions in neighboring skeletal muscle.     

Retroviral-mediated transfer and expression of the common gamma chain into human hematopoietic progenitors

The common gamma chain (gammac) of cytokine receptors is mutated in X-linked severe combined immunodeficiency, a lethal disorder characterized by the absence of both humoral and cellular immune defenses. Allogeneic bone marrow transplantation from HLA-identical siblings usually results in complete reconstitution of the immune system and is the current treatment of choice. Genetic correction and reinfusion of autologous hematopoietic stem cells represents an alternative therapeutic approach for those patients who lack suitable marrow donors. In this study, we show that retroviral-mediated transfer of the gammac gene results in efficient expression in CD34+ cells and high transduction rate of colony-forming progenitors.     

Hemopoietic marrow function in chronic neutropenia of blacks: cure of aplastic anemia by allogeneic marrow transplantation from a neutropenic sibling donor

A black patient with severe aplastic anemia is described who underwent successful bone marrow transplantation from a sibling with chronic neutropenia. During an evaluation to identify a suitable donor, it was found that the majority of family members tested had neutropenia, with no familial history of significant infections or related hospitalizations. In vitro hemopoietic culture studies of marrow from the patient's HLA-MLC-matched siblings showed normal numbers of pluripotential and committed hemopoietic progenitors; in vitro hemopoietic colony formation from the patient was markedly subnormal, consistent with the clinical picture of severe aplastic anemia. Following appropriate conditioning therapy, marrow transplanted from one of these neutropenic sibs produced full hematopoietic reconstitution. Posttransplant marrow culture studies of the patient showed restoration of a normal pattern of in vitro hemopoiesis. The in vitro culture studies and clinical experience in this patient support the concept that chronic neutropenia of blacks is not primarily a marrow progenitor cell disorder but, more likely, a manifestation of a genetically determined alteration in granulocyte kinetics.     

Long-term marrow reconstitutive ability of autologous grafts in lymphoma patients using peripheral blood mobilized with granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor compared to bone marrow

OBJECTIVES: The aim of this study was designed to compare the in vivo long-term hematopoietic potential of bone marrow and peripheral blood grafts. MATERIALS AND METHODS: Marrow progenitor cell recovery was assessed for up to 4 years in 227 patients. One hundred patients were treated for malignant lymphomas by autologous bone marrow transplantation (BMT) and 127 by peripheral blood progenitor cell transplantation (PBPCT). RESULTS: Marrow progenitor cell counts were decreased for several years with both bone marrow and peripheral blood grafts. They were not different according to the origin of the graft, despite the reduced duration of peripheral blood cell recovery observed after PBPCT. Granulocyte colony-stimulating factor (G-CSF) used for PB graft mobilization and after transplantation resulted in faster neutrophil recovery compared to granulocyte-macrophage colony-stimulating factor (GM-CSF) with no evidence of decreased marrow progenitor cell recoveries. On the other hand, postgraft administration of GM-CSF enhanced long-term colony-forming unit granulocyte-macrophage reconstitution only after BMT. Factors that influenced marrow progenitor cell reconstitution have been identified by univariate and multivariate analysis: age, gender, type of lymphoma, and postgraft administration of hematopoietic growth factors (HGF) for the whole patient group; gender, graft progenitor cell yields, and type of HGF (G-CSF vs GM-CSF) for the PBPCT group; and only type of HGF for the BMT group.Despite faster peripheral blood cell recovery, persistent deficiency of marrow progenitor cells was found several years after PBPCT, as observed after BMT. G-CSF-mobilized PBPCT resulted in faster neutrophil recovery compared to GM-CSF mobilization, with no difference in long-term hematopoietic reconstitution.     

Polyclonal reconstitution of human marrow after allogeneic bone marrow transplantation

Clonal dominance suggestive of reconstitution of marrow from small numbers of pluripotent hematopoietic stem cells has been noted in different experimental and clinical situations. Recipients of human allogeneic marrow transplants have not been previously studied to determine if clonal dominance occurs in this clinical setting. Clonal analysis of 20 allogeneic marrow transplant recipients was performed on DNA from peripheral blood neutrophils using restriction fragment length polymorphisms on the X chromosome. Similar studies were performed on 16 of the donors. To analyze the results further, recipients were paired with their respective donors. There was no evidence of shifts in cell populations contributing to the X chromosome inactivation patterns in recipient marrow grafts when compared with their respective donors. A mathematical model based on binomial statistics was adapted to estimate the numbers of reconstituting pluripotent hematopoietic stem cells. There was no evidence of clonal dominance suggestive of oligoclonal reconstitution in marrow grafts after allogeneic marrow transplantation. This does not preclude the possibility of oligoclonal reconstitution in other marrow transplant settings such as autologous transplantation.     

Eosinophilic fasciitis after allogeneic bone marrow transplantation

We describe a patient with eosinophilic fasciitis (EF) developing 8 months after an allogeneic bone marrow transplantation for acute myeloblastic leukemia. The patient responded to low dose prednisone. A full thickness skin-muscle-fascia biopsy detected the characteristic fascial changes of EF and distinguished it from other forms of chronic graft-versus-host-disease (GVHD). This distinction may be important since EF after bone marrow transplantation may occur more often and it may respond to treatment with low doses of prednisone whereas chronic GVHD usually requires more extensive immunosuppressive treatment.     

Assessment of the composition of bone marrow prior to and following autologous BMT and PBSCT by magnetic resonance

 Lumbar bone marrow was assessed by means of magnetic resonance (MR) in 23 examinations of eight patients who underwent autologous bone marrow transplantation (ABMT) or peripheral blood stem cell transplantation (PBSCT). Various imaging and spectroscopic techniques were applied for measurements carried out prior to conditioning for ABMT/PBSCT and in the course of reconstitution and correlated with clinical and blood chemistry data in these patients. The signal intensity from lumbar bone marrow was determined in T₁-weighted and water- and fat-selective MR images. The distribution of the magnetic field was demonstrated by a field-mapping method. Localized proton spectroscopy was performed from volume elements of 2 ml located in the central region of vertebral bodies in order to evaluate the fraction of the water signals, the transverse relaxation times T₂ of the signals from water and lipids, and the line widths of the spectral signals. Regions of bone marrow after inflammatory conditions or intensive irradiation are shown to be not involved in marrow reconstitution. Additional information about marrow composition was obtained by the magnetic field mapping and by the line widths in the spectra. Considerable alterations of the amount of paramagnetic hemosiderin were revealed following transplantation. Patients with low water signal and strong local inhomogeneities of the magnetic field in the marrow prior to transplantation had a delayed hematopoietic reconstitution compared with the patients lacking these MR features. Received: 9 November 1995 / Accepted: 11 March 1996     

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells

Blood cell transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho^+/++ (dull/bright) and Rho⁻ (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho^+/++ cell population contained >99% of committed progenitor cells with in vitro colony-forming ability. The Rho⁻ cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho⁻ stem cells or purified Rho⁻ stem cells in combination with large numbers of Rho^+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho⁻ stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.     

Acquired amegakaryocytic thrombocytopenia purpura and eosinophilic fasciitis: a long relapsing and remitting course

Acquired amegakaryocytic thrombocytopenia purpura (AATP) is a rare disorder of unclear etiology characterized by severe thrombocytopenia, preservation of erythroid and myeloid cell lines, and absence of megakaryocytes in the bone marrow. We report herein a patient who developed eosinophilic fasciitis preceding a diagnosis of AATP. Longitudinal follow-up and treatment of this individual show a relapsing and remitting disease course which appears closely related to the dosing of cyclosporine. Later in the treatment course, dosing of anti-thymocyte globulin (ATG) appeared to have important beneficial contributions in the management of this patient's disease.     

Rapid immune reconstitution and dendritic cell engraftment post-bone marrow transplantation with heterogeneous progenitors and GM-CSF treatment

OBJECTIVE: Bone marrow/hematopoietic stem cell transplantation (BMT) has been the treatment of choice for severe hematological diseases and cancers. Rapid host immune recovery following BMT is critical for reducing complications and improving therapeutic outcome. Here we report manipulations that facilitate rapid immune and dendritic cell (DC) reconstitution post-BMT for improvement in therapeutic outcome of BMT-based disease treatment. METHODS: Using lentiviral vector-modified or unmodified murine hematopoietic stem cells, we examined the engraftment efficiency and kinetics in immune reconstitution of unfractionated bone marrow cells (BM), lineage marker-negative (Lin-) hematopoietic progenitor cells (HPC), or purified Lin-Sca-1+ hematopoietic stem cells (HSC) at an equal hematopoietic progenitor number. RESULTS: Our study revealed that BM reconstituted host primary and secondary lymphoid tissues more efficiently and rapidly. Moreover, in a competitive BMT setting using lentiviral vector-engineered BM and HSC expressing GFP or DsRed respectively, we showed that GM-CSF treatment further enhanced DC reconstitution to therapeutic relevant level as early as 2 weeks post-BMT. On the other hand, Flt3 ligand was less effective in enhancing DC reconstitution till 3 weeks post-BMT. This accelerated DC engraftment by GM-CSF treatment correlated well with improved overall immune reconstitution and enhanced activation of antigen-specific T cells post-BMT. CONCLUSION: This study suggests that use of heterogeneous BM for transplantation facilitates more rapid immune reconstitution, especially in the presence of DC-stimulating cytokines. This improved immune reconstitution would provide additional therapeutic benefits for BMT-based immunotherapy and gene therapy of genetic disorders and cancers.     

Eosinophilic fasciitis associated with autoimmune phenomena after bone marrow transplantation: report of two cases

Between January 1992 and May 2004, 189 patients underwent allogeneic bone marrow transplantation (BMT) for haematological malignancies from HLA-identical sibling donors in our transplantation unit. Of the 189 patients, 2 developed eosinophilic fasciitis (EF). The first patient developed Hashimotos thyroiditis and EF 11 and 21 months after BMT, respectively. In the second patient EF occurred 9 months after BMT, accompanied by antinuclear antibodies, antiextractable nuclear antigens and antigliadin antibodies. Both patients were treated with extracorporeal photochemotherapy (ECP), resulting in improvement of fasciitis in both and normalization of antithyroid antibodies in the first patient. Our data confirm the rarity of fasciitis after BMT and the efficacy of ECP, recently applied experimentally in one patient for the treatment of fasciitis after BMT. Moreover, we report for the first time the association of fasciitis with autoimmune phenomena after BMT. The correlation between the two entities is supported by remission of Hashimotos thyroiditis after ECP treatment for fasciitis.     

Rheumatoid arthritis with eosinophilic fasciitis and pure red cell aplasia

A case of longstanding rheumatoid arthritis with concurrent development of eosinophilic fasciitis and pure red cell aplasia is described. A simultaneous occurrence of all 3 disorders has not been reported. Treatment with moderate dosages of prednisone resulted in a prompt and complete remission of both the fasciitis and the selective marrow aplasia.     

Homing and engraftment defects in ex vivo expanded murine hematopoietic cells are associated with downregulation of beta1 integrin.

OBJECTIVE: Hematopoietic progenitors generated by ex vivo expansion "home" less efficiently to the bone marrow (BM) after intravenous transplantation than fresh cells. To explore the underlying cause of this transplantation defect, we examined the homing and engraftment properties in vivo of fresh and cultured marrow cells differing in beta1 integrin expression. MATERIALS AND METHODS: Fresh murine BM cells, or the expanded progeny of enriched Sca-1(+) c-kit(+)Lin(-) stem cells, were fractionated into beta1(-/lo) and beta1(+) subpopulations by cell sorting. These populations were assayed for their content of in vitro colony-forming cells (CFCs), cells able to provide radioprotection, and early and long-term multilineage hematopoietic reconstitution following transplantation into myeloablated recipients. These endpoints were correlated with the homing properties of beta1(-/lo) and beta1(+) cells that were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) and tracked to hematopoietic organs 24 hours after injection into lethally irradiated mice. RESULTS: Most normal stem and progenitor cells express high levels of beta1 integrin. In contrast, most clonogenic cells generated in vitro are beta1(-/lo). Consequently, expanded beta1(-/lo) progenitors failed to provide radioprotection or repopulate the hematopoietic system following intravenous transplantation. Defective engraftment of expanded cells was associated with reduced homing of beta1(-/lo) cells to the bone marrow. CONCLUSION: Downregulation of beta1 integrin on primitive hematopoietic cells during ex vivo expansion reduces their homing efficiency and negatively impacts hematopoietic reconstitution in vivo. Strategies directed at preserving beta1 integrin expression during culture may improve the clinical utility of expanded hematopoietic cells.     

Dysregulated Mpl-Ligand production by hemopoietic cellsinduces a fatal myeloproliferative syndrome in mice

To evaluate the effects of long-term high-dose exposure to Mpl-ligand also called thrombopoietin (TPO), C57BL/6J murine marrow cells were infected with a retrovirus carrying the murine TPO gene. Mice were treated 4 days by 5-FU and marrow cells were then infected by coculture using a MPZen vector containing the murine TPO cDNA. Non adherent marrow cells were transplanted into lethally irradiated recipients. A majority of hematopoietic cells in the marrow, spleen, thymus and blood was transduced by the retroviral vector, one and three months after reconstitution. Plasma TPO activity in transplanted mice was extremely high (10⁴ U/ml). A disease with two distinct steps was observed. During the two first months after transplantation, platelet (plt) and white blood cell (WBC) counts increased 4- and 10-fold, respectively. Abnormal platelet size and granules were observed. Spleen weight increased 4-fold and marrow cellularity decreased 5-fold. Histology revealed hyperplasia of the megakaryocytic and myeloid cells. Total numbers of CFU-MK and CFU-GM increased. In contrast, the hematocrit progressively fell accompanied by a decrease in the erythroblasts and CFU-E numbers. Beginning two months after transplantation, plt and WBC numbers also declined. Thrombocytopenia was noted 5 months after transplantation. The Hcts continued to decrease. Few cells were isolated from the marrow cavities and spleens. Histology revealed fibrosis of the marrow and spleen and significant osteosclerosis of the marrow. An extramedullary hematopoiesis was observed in numerous organs such as the liver or the kidney. Total numbers of progenitors were very low in hematopoietic organs. Mice died 7 months after transplantation with severe pancytopenia. Two early deaths were observed with a marked increase in blast cells. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described [Yan et al (1995) Blood 86: 4025]. In conclusion, dysregulated TPO production by hemopoietic cells in mice results in a fatal myeloproliferative disease which mimics the clinical evolution of idiopathic myelofibrosis observed in man.     

Late Relapse of Acute Myelogenous Leukemia Followed by Epstein-Barr Virus—Associated Lymphoproliferative Disease 11 Years After Allogeneic Bone Marrow Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) following myeloablative conditioning represents the treatment of choice for patients with chemotherapy-resistant leukemia. We describe a 49-year-old man with advanced, refractory acute myelogenous leukemia (AML) that was treated successfully by allogeneic bone marrow transplantation from a sibling donor with HLA mismatched at 1 locus. However, the patient developed a quiescent form of chronic graft-versus-host disease (GVHD) 7 years after transplantation, requiring long-term immunosuppressive therapy. AML relapse was documented 11 years after transplantation. Subsequently, Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorder (PTLD) was also diagnosed. Immune reconstitution after allo-HSCT might have been impaired by the persistent chronic GVHD and the prolonged administration of immunosuppressive agents. As a result, immune surveillance against remaining quiescent leukemic cells as well as viral infection may have been defective, leading to the relapse of leukemia and EBV-associated PTLD.     

Case Report of Unilateral Eosinophilic Fasciitis in a Vietnamese Woman

Symmetric skin thickening of the limbs with deep fascial inflammation is the hallmark of eosinophilic fasciitis. We describe a woman who presented with unilateral progressive skin thickening. Examination of a full-thickness skin biopsy revealed an inflammatory process and fascial changes consistent with eosinophilic fasciitis. In contrast to other scleroderma mimics, eosinophilic fasciitis generally responds rapidly to glucocorticoid therapy. It is possible that unilateral eosinophilic fasciitis is under-recognized and can easily be misdiagnosed as another scleroderma variant if a full-thickness biopsy is not reviewed by a dermatopathologist. Recognition of this subtype of eosinophilic fasciitis is important given the profound differences in prognosis of eosinophilic fasciitis and other scleroderma variants.     

Homeostasis of telomere length rather than telomere shortening after allogeneic peripheral blood stem cell transplantation

Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell-depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.     

Enhanced reconstitution of hematopoietic organs in irradiated mice, following their transplantation with bone marrow cells pretreated with recombinant interleukin 3

Lethally irradiated C3H/eb mice were injected with syngeneic bone marrow cells that had been exposed for 4 h in vitro to purified bacterially synthesized interleukin 3 (rIL-3). Control mice were injected with cells exposed to incubation medium only. Mice injected with rIL-3-treated cells exhibited, on day 10 after transplantation an 8.2-fold and 2.7-fold increase in number of myeloid progenitors in their spleen and bone marrow, respectively, but the in vitro differentiation pattern of the myeloid progenitors was not affected. There was, however, an increase in the number of cells per individual in vitro myeloid colony (CFU-C) of the rIL-3-treated mice. The latter mice also showed a 1.6-fold increase in the number of splenic colony-forming units (CFU-S), a higher self-renewal capacity of hematopoietic progenitors, and a higher number of leukocytes in the peripheral blood. These results indicate that the injection into lethally irradiated recipients of bone marrow cells briefly pretreated in vitro with rIL-3 significantly enhances the reconstitution of their hematopoietic organs, and suggest that the in vitro pretreatment of bone marrow cells with appropriate stimulating factors could be useful in bone marrow transplantation.