John D. Jackson

Rapid immunologic reconstitution following transplantation with mobilized peripheral blood stem cells as compared to bone marrow

A majority of patients with intermediate or high-grade non-Hodgkin’s lymphoma (NHL) who are treated with high-dose chemotherapy (HDT) and hematopoietic stem cell transplantation subsequently relapse. Until recently, transplantation was associated with high morbidity and mortality and the focus was on improving the safety of this procedure. However, the use of growth factors and other supportive measures has successfully reduced treatment mortality to less than 5%. Therefore, new strategies need to be developed to eliminate the growth of any occult tumor cells reinfused with the stem cell products and the tumor cells remaining in the patient. One approach is to improve the immune function of the patients by a more rapid immune reconstitution and augmentation of effector cell function. We report studies comparing immune recovery following HDT and autologous peripheral stem cell transplantation (PSCT) as compared to autologous bone marrow transplantation (ABMT). These studies examined patients with intermediate and high-grade non-Hodgkin’s lymphoma (NHL) who were treated with HDT and PSCT (n = 56) or ABMT (n = 60). The PSCT patients had a significantly faster recovery of circulating monocytes (CD14+ cells), natural killer ((NK) CD56+) cells, T helper (CD4+) cells, TCRγ/δ cells, and naive T lymphocytes (CD45RA+). Following ABMT there was a significantly more rapid increase in the frequency of T suppressor/effector (CD8+) cells, B (CD19+) cells, CD34+ cells, polymorphonuclear leukocytes (PMN) and memory T lymphocytes (CD45RO+). The CD4:CD8 and CD45RA:CD45RO ratios were consistently higher in the PSCT group as compared to ABMT suggesting an improved ratio of T helper to T effector/suppressor cells and naive T cells. The differences in cellular phenotype translated into improved T cell function (PHA mitogenesis) and T cell help (pokeweed mitogenesis). In addition, there was an accelerated reconstitution of NK cell activity following PSCT as compared to ABMT. The more rapid reconstitution of NK and T cells in patients rescued with PSCT as compared to ABMT may contribute to an improved clinical outcome. Further, patients receiving a PSCT may be more responsive to adjuvant immunotherapy following transplantation.

Hematopoietic recovery after allogeneic blood stem-cell transplantation compared with bone marrow transplantation in patients with hematologic malignancies.

PURPOSE To compare hematopoietic recovery, duration of hospitalization, and 100-day survival in patients who received allogeneic-blood stem cells (BSC) or conventional allogeneic bone marrow transplantation (BMT). PATIENTS AND METHODS From December 1994 to August 1995, 21 patients participated in a phase II study of allogeneic BSC transplantation. Cells mobilized with granulocyte colony-stimulating factor (G-CSF; 5 micrograms/kg/ d) were collected from human leukocyte antigen (HLA)-matched related donors and cryopreserved. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and methotrexate. G-CSF (10 micrograms/kg/d) was administered posttransplant. The outcomes were compared with 22 identically treated historical patients who received allogeneic BMT. RESULTS The median infused CD34+ cell and granulocyte-macrophage colony-forming unit (CFU-GM) content were 7.73 x 10(4)/kg and 41.6 x 10(4)/kg, respectively. The median time to a neutrophil count greater than 500/ microL was 11 days after BSC and 16.5 days after BMT (P = .0003). A trend toward faster platelet and RBC recovery after BSC was observed. BSC patients received fewer platelet transfusions: 10 versus 19 (P = .015). The median length of hospitalization was shorter after BSC transplantation: 25 versus 31.5 days (P = .0243). The 100-day survival rates were similar: 83% after BSC and 75% after BMT (P = .3585). The incidence of acute GVHD grade II to IV was 57% and 45% for BSC and BMT, respectively (P = .4654). CONCLUSION In comparison to BMT, allogeneic BSC transplantation may result in faster hematopoietic recovery, shorter hospital stay, and similar early survival. Whether allogeneic BSC are superior to bone marrow needs to be determined in randomized trials.

Changes in human bone marrow fat content associated with changes in hematopoietic stem cell numbers and cytokine levels with aging

Hematological deficiencies increase with aging, including anemias, reduced responses to hematopoietic stress and myelodysplasias. This investigation tested the hypothesis that increased bone marrow (BM) fat content in humans with age was associated with decreased numbers of side population (SP) hematopoietic stem cells, and this decrease correlated with changes in cytokine levels. BM was obtained from the femoral head and trochanteric region of the femur removed at surgery for total hip replacement (N = 100 subjects). In addition, BM from cadavers (N = 36), with no evidence of hip disease, was evaluated for fat content. Whole trabecular marrow samples were ground in a sterile mortar and pestle, and cellularity and lipid content determined. Marrow cells were stained with Hoechst dye and SP profiles were acquired. Plasma levels of insulin‐like growth factor (IGF)‐1, stromal‐derived factor (SDF)‐1 and interleukin (IL)‐6 were measured using ELISA. Fat content in the BM of human subjects and cadavers increased with age. The numbers of SP stem cells in BM as well as plasma IGF‐1 and SDF‐1 levels decreased in correlation with increased BM fat. IL‐6 had no relationship to changes in marrow fat. These data suggest that increased BM fat may be associated with a decreased number of SP stem cells and IGF‐1 and SDF‐1 levels with aging. These data further raise a more general question as to the role of adipose cells in the regulation of tissue stem cells.

Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: Correlation with cytokines

Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive.

Allogeneic-blood stem-cell collection following mobilization with low-dose granulocyte colony-stimulating factor.

PURPOSE The optimal dose of granulocyte colony-stimulating factor (G-CSF) for mobilization of allogeneic-blood stem cells (AlloBSC) has yet to be determined. As part of a prospective trial, 41 related human leukocyte antigen (HLA)-matched donors had blood cells mobilized with G-CSF at 5 micrograms/kg/d by subcutaneous administration. The purpose of this trial was to monitor adverse effects during G-CSF administration and stem-cell collection, to determine the optimal timing for stem-cell collection, and to determine the cellular composition of stem-cell products following G-CSF administration. PATIENTS AND METHODS The median donor age was 42 years. Apheresis began on day 4 of G-CSF administration. At least three daily 12-L apheresis collections were performed on each donor. A minimum of 1.0 x 10(6) CD34+ cells/kg (recipient weight) and 8.0 x 10(8) mononuclear cells/kg were collected from each donor. All collections were cryopreserved in 5% dimethyl sulfoxide and 6% hydroxyethyl starch. RESULTS Toxicities associated with G-CSF administration and the apheresis process included myalgias/arthralgias (83%), headache (44%), fever (27%), and chills (22%). The median baseline platelet count of 242 x 10(4)/ mL decreased to 221, 155, and 119 x 10(6)/mL on days 4, 5, and 6 of G-CSF administration, respectively. Median numbers of CD34+ cells in collections 1, 2, and 3 were 1.99, 2.52, and 3.13 x 10(6)/kg, respectively. The percentage and total number of CD4+, CD8+, and CD56+/CD3- cells remained relatively constant during the three collections. Median total numbers of cells were as follows: CD34+, 7.73 x 10(6)/kg; and lymphocytes, 6.93 x 10(8)/kg. CONCLUSION Relatively low doses of G-CSF can mobilize sufficient numbers of AlloBSC safely and efficiently.

Enhanced re-endothelialization of acellular kidney scaffolds for whole organ engineering via antibody conjugation of vasculatures

Decellularization of whole organs, such as the kidney hold great promise in addressing donor shortage for transplantation. However, successful implantation of engineered whole kidney constructs has been challenged by the inability to maintain endothelial cell coverage of the vasculature matrix, resulting in excessive blood clots, loss of vascular patency, and cell death within the construct. In this study, we describe an endothelial cell seeding approach that permits effective coating of the vascular matrix of the decellularized porcine kidney scaffold using a combination of static and ramping perfusion cell seeding. Furthermore, conjugation of CD31 antibodies to the vascular matrix improved endothelial cell retention on the vasculatures, which enhanced vascular patency of the implanted scaffold. These results demonstrate that our endothelial cell seeding method combined with antibody conjugation improves endothelial cell attachment and retention leading to vascular patency of tissue-engineered whole kidney in vivo.

Phase I trial of recombinant fusion protein PIXY321 for mobilization of peripheral-blood cells.

PURPOSE Mobilization of peripheral-blood cells (PBC) with cytokines alone results in rapid hematopoietic recovery and avoids the potential morbidity associated with mobilization by chemotherapy. PIXY321, a fusion protein that consists of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3), has enhanced hematopoietic colony-forming activity as compared with individual or equimolar combinations of the two cytokines. A phase I trial of PIXY321 for mobilization of PBC in patients with malignant lymphoma was performed. PATIENTS AND METHODS Thirteen patients with malignant lymphoma who were eligible for high-dose therapy (HDT) were enrolled onto the trial. All patients were ineligible for autologous bone marrow transplantation due to overt metastatic disease in the marrow or to severe marrow hypocellularity. PIXY321 was administered at three dose levels of 250, 500, and 750 micrograms/m2/d by continuous infusion until completion of PBC collections. Collections were initiated when the WBC count was greater than 10 x 10(9)/L or 4 days after the initiation of PIXY321, whichever came first. Collections were continued until a minimum of 6.5 x 10(8) mononuclear cells (MNC)/kg patient weight were obtained. RESULTS PIXY321 was generally well tolerated. Side effects associated with PIXY321 administration did not exceed grade 2 and included fever (85%), chills/sweats (54%), myalgias (38%), fatigue (31%), nausea/vomiting (31%), headache (31%), edema (23%), and rhinorrhea (23%). The median numbers of colony-forming units-granulocyte/macrophage (CFU-GM) in the graft products for the three dose levels were 0.31, 2.94, and 2.88 x 10(4)/kg, respectively; the median numbers of burst-forming units-erythroid (BFU-e) were 0.20, 6.94, and 12.78 x 10(4)/kg, and the median numbers of CD34+ cells were 2.30, 0.74, and 0.39 x 10(6)/kg. Following transplantation, the median times to an absolute neutrophil count (ANC) > 0.5 x 10(9)/L were 12, 15, and 12 days, respectively, and the median times to platelet transfusion independence were 30, 19, and 15 days. CONCLUSION PIXY321 can be safely administered and effectively mobilizes PBC in patients with bone marrow defects. PIXY321-mobilized PBC autotransplants result in rapid and sustained hematopoietic recovery.

Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen.

Hematopoietic chimerism has been used in the laboratory to induce life-long immunologic tolerance to donor antigens. The present study demonstrates that mice transplanted with autologous bone marrow cells retrovirally transduced to express HLA-A2.1 develop a significantly depressed immune response to this antigen while retaining normal reactivity to HLA-B7. Retrovirus-mediated transduction was performed using whole bone marrow-producer cell coculture. This approach did not result in significant gene transfer into hematopoietic progenitor cells. Despite this, the antibody response to HLA-A2.1 in mice reconstituted with genetically modified BMC was completely suppressed three months following bone marrow transplantation. Cell-mediated immunity to HLA-A2.1 was partially suppressed in three-fourths of animals tested three months later, although one animal had a CTL profile similar to that an of HLA-A2.1 transgenic mouse. Complete suppression of the antibody-mediated immune response occurred when only one-third of mice had evidence of the introduced genes in their spleen and one-tenth had the introduced sequences in their circulating WBCs by PCR. In conclusion, engineering of BMC to express donor MHC genes may be an alternative to xenogeneic BMT to induce chimerism and tolerance. More efficient transduction of bone marrow progenitor cells may result in more persistent gene expression and long-lasting transplantation tolerance in recipients of genetically modified bone marrow. Successful application of this technology may also be useful in altering immune responses to other external and self antigens.

Inflammation Associated With Obesity: Relationship With Blood and Bone Marrow Endothelial Cells

The purpose of this study was to assess the inflammatory nature of obesity and its effect on blood and bone marrow endothelial cell populations. Obese patients (BMI ≥30) had significantly higher concentrations of the inflammatory marker C‐reactive protein (CRP) (P = 0.03) and lower concentrations of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) (P = 0.05). This cytokine profile is consistent with obesity being an inflammatory condition and is further supported by the significant correlation between total white blood cell count and BMI (r = 0.15; P = 0.035). High BMI was associated with significantly lower numbers of early endothelial cells (CD45−/CD34+) in the bone marrow (r = −0.20; P = 0.0068). There was also a significant inverse correlation between BMI and a more mature endothelial cell phenotype (CD45−/31+) in the blood (r = −0.17; P = 0.02). In addition, there was a significant correlation between BMI‐ and endothelial‐related cells of hematopoietic origin (CD133+/VEGFR‐2+) in the bone marrow (r = −0.26; P = 0.0007). Patients with higher plasma IL‐10 and insulin‐like growth factor (IGF) concentrations had higher numbers of endothelial phenotypes in the bone marrow suggesting a protective effect of these anti‐inflammatory cytokines. In conclusion, this work confirms the inflammatory nature of obesity and is the first to report that obesity is associated with reduced endothelial cell numbers in the bone marrow of humans. These effects of obesity may be a potential mechanism for impaired tissue repair in obese patients.

Differential recovery of polymorphonuclear neutrophils, B and T cell subpopulations in the thymus, bone marrow, spleen and blood of mice following split-dose polychemotherapy

In these studies, we examined the effect of a maximum-tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea carmustine on neutrophil and lymphocyte subpopulations in the peripheral blood (PBL), thymus, bone marrow and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation for breast cancer, suppressed both B and T cell populations and T cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. In the peripheral blood, 7 days following initiation of chemotherapy, there was a twofold increase in the percentage of granulocytes as compared to the level in control animals on the basis of a differential count. The polymorphonuclear neutrophil (PMN) frequency in the bone marrow was increased on day 14 and statistically identical to that in control mice on all other days analyzed. In contrast to the bone marrow cells and PBL on day 7, the frequency of PMN in the spleen and thymus was depressed. B cells (B220+) were depressed in the PBL, spleen and bone marrow and took 18–32 days to return to their normal frequency, while the frequency of B cells in the thymus was increased owing to a loss of immature T cells. The percentage of CD3+ cells in the thymus, spleen and bone marrow was significantly increased and required 10–18 days to return to normal levels, while the absolute number of CD3+ cells in the blood varied around the normal value. The ratio of CD4+ to CD8+ cells in all the organs studied varied only slightly owing to a similar reconstitution of CD4+ and CD8+ cells. In contrast to the phenotypic recovery of the CD3+, CD4+ and CD8+ cells, the ability of the splenic lymphocytes to respond to concanavalin-A was depressed and remained depressed, despite the phenotypic reconstitution of the T cell subsets, on the basis of both percentage and absolute cell number. These results show a selective T and B cell depression following multi-drug, split-dose chemotherapy in tissue and blood leukocyte populations and a chronic depression in T cell function.