Generation of mouse models for type 1 diabetes by selective depletion of pancreatic beta cells using toxin receptor-mediated cell knockout.

By using the toxin receptor-mediated cell knockout (TRECK) method, we have generated two transgenic (Tg) murine lines that model type 1 (insulin-dependent) diabetes. The first strain, C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg, carries the diphtheria toxin receptor (hDTR) driven by the human insulin gene promoter, while the other strain, C57BL/6-ins2(BAC)-TRECK-Tg, expresses hDTR cDNA under the control of the mouse insulin II gene promoter. With regard to the C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg strain, only one of three Tg strains exhibited proper expression of hDTR in pancreatic ß cells. By contrast, hDTR was expressed in the pancreatic ß cells of all four of the generated C57BL/6-ins2(BAC)-TRECK-Tg strains. Hyperglycemia, severe ablation of pancreatic ß cells and depletion of serum insulin were observed within 3days after the administration of diphtheria toxin (DT) in these Tg mice. Subcutaneous injection of a suitable dosage of insulin was sufficient for recovery from hyperglycemia in all of the examined strains. Using the C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg model, we tried to perform regenerative therapeutic approaches: allogeneic transplantation of pancreatic islet cells from C57BL/6 and xenogeneic transplantation of CD34(+) human umbilical cord blood cells. Both approaches successfully rescued C.B-17/Icr-Prkdc(scid)/Prkdc(scid)-INS-TRECK-Tg mice from hyperglycemia caused by DT administration. The high specificity with which DT causes depletion in pancreatic ß cells of these Tg mice is highly useful for diabetogenic research.

Quality-assessments of characteristics of placental/umbilical cord blood associated with maternal age- and parity-related factor.

Umbilical cord blood (CB) has been widely used for unrelated allogeneic stem cell transplantation. It is important to determine the quality of CB units to avoid frequent problem of limited cell yields. However, no practical and/or optimum obstetric factors to predict them are yet available. This study analyzed the relationship between maternal/neonatal obstetric factors and the laboratory parameters of CB units to identify the optimum factors associated with a high yield of total nucleated cells (TNC). Primiparae in their early 30s may be one of the first selection criteria for CB donors to obtain higher yield of TNC.

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue.

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9) cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.

The current status of umbilical cord blood collection in Japanese medical centers: survey of obstetricians.

As the first step of UCB banking, UCB collection has an important role in banking procedures. The aim of this study was to reveal the current status of UCB collection and discuss the management of the UCB bank. We conducted a questionnaire survey at medical centers collecting UCB, followed by semi-structured interviews with some respondents. Out of 38 institutes, 11 respondents (28.9%) thought that collection of UCB in addition to their routine medical services puts a burden on physicians. The obstetricians involved in the UCB collection are generally willing to participate in the procedure under current circumstances at medical institutes.

Concise review: isolation and characterization of cells from human term placenta: outcome of the first international Workshop on Placenta Derived Stem Cells.

Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy-based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23-24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.

Maternal and neonatal factors associated with the high yield of mononuclear low-density/CD34+ cells from placental/umbilical cord blood.

Placental/umbilical cord blood (CB) contains nucleated cells and hematopoietic stem/progenitor cells (CD34(+) cells). However it is difficult to predict the number of nucleated/CD34(+) cells in each CB before cell processing. Despite many previous studies from institutes affiliated with CB banks in metropolitan areas, little information is available regarding the characteristics of CB units from other medical facilities. The purpose of the present study was to analyze the maternal/neonatal factors on the yield of cells in CB units. A total of 176 CB units were obtained from single-birth and normal vaginal deliveries. Mononuclear low-density (LD) cells were separated using Ficoll-Paque within 24 hrs after CB collection and then processed for the purification of CD34(+) cells. A multiple linear regression analysis was performed to assess the correlations between the yield of cells and maternal/neonatal factors including maternal age, gravid status, duration of labor, gestational age, neonatal height and weight, cord length, and meconium in the amniotic fluid. The total LD cells per CB unit had a weak positive correlation with the maternal age of primigravidae. The total LD cells per CB unit from the primigravidae aged > or = 25 were significantly higher than those from the primigravidae aged < or = 24. The total CD34(+) cells per CB unit from the 1-gravidae were significantly higher than those from the 2-gravidae and 3-gravidae, respectively among all donors. These results indicate that the CB units from the primigravidae aged > or = 25 are more likely to contain higher yield of LD/CD34(+) cells.

Results of a phase I clinical study using dendritic cell vaccinations for thyroid cancer.

OBJECTIVE: We assessed the feasibility and efficacy of dendritic cell (DC) therapy for advanced thyroid papillary and follicular cancer. DESIGN: Six Japanese patients (2 men and 4 women; aged 46-72 years, mean 60 years), who were diagnosed as advanced thyroid cancer with refractory distant metastases (papillary, n=5; follicular, n=1), were enrolled. Patients were first vaccinated weekly for 4 weeks with 10(7) autologous tumor lysate-pulsed monocyte-derived mature DCs followed by fortnightly vaccinations for 8 weeks (total=8 vaccinations). Lowdose (350 KIU) interleukin-2 was also administered for 3 days at each vaccination. Clinical response, adverse effects, delayed-type hypersensitivity skin testing (DTH), and IFN-( ) production by peripheral CD3(+) lymphocytes were evaluated. MAIN OUTCOME: Of the 6 patients, disease was assessed as stable in 2 and as progressive in 4. No adverse events were observed. Results of DTH and IFN-( ) production in peripheral lymphocytes did not correlate to the clinical response. CONCLUSIONS: DC immunotherapy could be administered to patients with thyroid papillary or follicular cancer without substantial side effects.

The effects of glycosaminoglycans on thrombopoietin-induced megakaryocytopoiesis.

BACKGROUND AND OBJECTIVES: The extracellular matrix plays an essential role in normal hematopoiesis. Proteoglycans and glycosaminoglycans (GAG) are major components of the matrix. In this study, the effects of various GAG on the proliferation and differentiation of CD34+ megakaryocytic progenitor cells (CFU-Meg) were evaluated in vitro. DESIGN AND METHODS: CD34+ cells were highly purified from steady-state human peripheral blood. The GAG tested were hyaluronic acid (from humans, pigs and roosters), keratan sulfate, heparan sulfate, chondroitin sulfate (from whale, shark or squid cartilage) and dermatan sulfate (DS). RESULTS: When used alone, none of the GAG supported the clonal growth of CFU-Meg; however, in cultures stimulated by recombinant human thrombopoietin, human hyaluronic acid, whale chondroitin sulfate and DS significantly enhanced such growth. In particular, the addition of DS resulted in increases of about 1.3-fold, 1.6-fold and 2.0-fold in the numbers of total cells, megakaryocytes and CFU-Meg, respectively, compared with the control culture stimulated by thrombopoietin alone after 9-12 days of serum-free liquid culture. Furthermore, DS induced the generation of hyperploid megakaryocytes and promoted pro-platelet formation. Chemical fragmentation and desulfation of DS showed that a chain of at least 12 saccharides is required for colony-promoting activity and that the sulfate groups play an essential role. INTERPRETATION AND CONCLUSIONS: DS acts on an immature population of CD34+ cells, stimulates the proliferation of CFU-Meg, and enhances the terminal maturation of megakaryocytes and thrombopoiesis. These results suggest that DS has a wide spectrum of action in promoting megakaryocytopoiesis and thrombopoiesis.

Regeneration of megakaryocytopoiesis and thrombopoiesis in vitro from X-irradiated human hematopoietic stem cells.

In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34(+) megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34(+) cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41(+) particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34(+) cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations.

Carbonic anhydrase II is a tumor vessel endothelium-associated antigen targeted by dendritic cell therapy.

Tumor-associated antigens are promising candidates as target molecules for immunotherapy and a wide variety of tumor-associated antigens have been discovered through the presence of serum antibodies in cancer patients. We previously conducted dendritic cell therapy on 10 malignant melanoma patients and shrinkage or disappearance of metastatic tumors with massive necrosis occurred in two patients. In this study, we found a 29-kDa protein against which antibody was elicited by dendritic cell therapy in one of the two patients. Matrix-assisted laser desorption ionization-time of flight/mass spectrometry analysis of the protein isolated by two-dimensional electrophoresis combined with Western blots revealed that the 29-kDa protein was carbonic anhydrase II (CA-II). Immunohistochemistry of the tumors and normal tissues showed that CA-II was expressed in the tumor vessel but not in normal vessel endothelium. CA-II expression in tumor endothelium was observed as well in other cancers including esophageal, renal, and lung cancers. In an in vitro angiogenesis model, CA-II expression of normal human vein endothelial cells was significantly up-regulated when cells were cultured in the acidic and hypoxic conditions indicative of a tumor environment. These findings suggest that CA-II is a tumor vessel endothelium-associated antigen in melanoma and other cancers, and elicitation of serum anti-CA-II antibody by dendritic cell therapy may be associated with good clinical outcome including tumor reduction.

Different radiosensitive megakaryocytic progenitor cells exist in steady-state human peripheral blood.

CD34 antigen is a novel marker for human hematopoietic stem/progenitor cells. In the present study, two cell fractions, CD34low and CD34high, were prepared from steady-state human peripheral blood on the basis of CD34 antigen expression. The colony-forming unit megakaryocytes (CFU-Meg) contained in each cell fraction were compared for X-radiation sensitivity and cytokine action. The content of CD34+CD45+ cells in the CD34low and CD34high cell fractions was 74.8% and 88.8%, respectively, and the frequency of thrombopoietin (TPO)-supported CFU-Meg in the CD34low cell fraction was 1.9 times higher than that in CD34high. The CFU-Meg in CD34high were more radiosensitive than those in CD34low, indicating that steady-state human peripheral blood contains different types of CFU-Meg. However, no significant differences were observed between cell fractions in the radiation survival curves of CFU-Meg stimulated by TPO plus cytokines except granulocyte colony-stimulating factor (G-CSF). TPO plus interleukin 3 was the optimal combination for survival of both types of CFU-Meg after X irradiation. The present study also demonstrated that TPO plus G-CSF is able to increase the survival of irradiated CD34low CFU-Meg. These results suggest that two megakaryocytic progenitor populations with different radiosensitivity and cytokine responses are found in steady-state human peripheral blood.

Fibroblast growth factor and ex vivo expansion of hematopoietic progenitor cells.

Fibroblast growth factor (FGF) belongs to a family of heparin-binding polypeptides and shows multiple functions including cell proliferation, differentiation, survival and motility. The expression of FGF receptors is widely distributed on different hematopoietic progenitor cells and stromal cells, and FGFs play an important role in hematopoietic stem cell homeostasis. FGFs have been shown to sustain the proliferation of hematopoietic progenitor cells, maintaining their primitive phenotype. Basic FGF (bFGF, FGF-2) stimulates the formation of an adherent stromal cell layer in human long-term bone marrow cultures, and promotes hematopoietic cell development. FGF-2 has also been shown to synergize with other hematopoietic growth factors to enhance in vitro colony formation by several classes of hematopoietic progenitor cells. Results of ex vivo expansion and clinical trials to date suggest that hematopoietic cells cultured under stroma-free cytokine combination conditions may be insufficient to restore hematopoiesis after a myeloablative conditioning regimen, although some recent trials demonstrated an improvement in engraftment and a reduction of the period of pancytopenia, especially neutrophils and platelets, after transplantation. A recent study by our group demonstrated that FGF-2 is effective in supporting the generation of megakaryocytic progenitor cells during ex vivo expansion. These observations could be useful in reducing the long period of severe thrombocytopenia that occurs frequently after umbilical/placental cord blood transplantation. The development of more effective amplifying systems for hematopoietic stem/progenitor cells can be expected since FGFs have multiple functions in hematopoiesis.

Differential expansion of umbilical cord blood mononuclear cell-derived natural killer cells dependent on the dose of interleukin-15 with Flt3L.

OBJECTIVE: We investigated the effect of interleukin-15 (IL-15) with Flt3 ligand (Flt3L) on the expansion and activation of NK cells derived from umbilical cord blood mononuclear cells (UCB-MNCs). MATERIALS AND METHODS: UCB-MNCs were cultured at 1 to 100 ng/mL of IL-15 + Flt3L (10 ng/mL) compared with 1 to 500 ng/mL of IL-2 + Flt3L (10 ng/mL). Cultured cells were assessed for surface marker and we calculated absolute number of NK cells and T cells. The cytotoxic activity was analyzed with purified NK cells. RESULTS: After 2 weeks culture with 5 ng/mL of IL-15 + Flt3L, the fold inductions of absolute number of NK cells significantly increased to 20.9-fold +/- 9.3-fold of the number of NK cells on day 0 (p < 0.05), with 24.4-fold +/- 16.1-fold of T cells. But with 50 ng/mL of IL-15 + Flt3L, fold induction of NK cells decreased to 5.1-fold +/- 3.9-fold, while T cells showed 34.8-fold +/- 18.7-fold (n = 8). The proportion of NK vs T cells showed to be significantly higher (1.61 +/- 0.91) with 5 ng/mL of IL-15 than with 50 ng/mL of IL-15 (0.12 +/- 0.03). Such proportional change of NK/T cells could not be observed with IL-2. Immunophenotypes of CD56, CD16, LFA1, CD94, CD8, and perforin of cultured NK cells with 10 ng/mL of IL-15 + Flt3L showed the same pattern of those with 50 ng/mL of IL-2 + Flt3L. Cytotoxic activity against K562 of cultured NK cells resulted in the same level as adult peripheral blood (PB)-derived NK cells. CONCLUSIONS: Higher induction of NK cells derived from UCB-MNCs was achieved by low dose (5 to 10 ng/mL) rather than high dose (> 50 ng/mL) of IL-15.

Ex vivo manipulation of umbilical cord blood-derived hematopoietic stem/progenitor cells with recombinant human stem cell factor can up-regulate levels of homing-essential molecules to increase their transmigratory potential.

OBJECTIVE: The cause of delayed hematopoietic reconstitution after umbilical cord blood transplantation (UCBT) remains controversial. We hypothesized that hematopoietic stem/progenitor cells (HS/PCs) from UCB have some defects of the homing-related molecules responsible for their slow engraftment. MATERIALS AND METHODS: A homing-related molecule repertoire expressed on HS/PCs from fresh and cryopreserved UCB, mobilized peripheral blood (mPB), and bone marrow (BM) were compared using sensitive, four-color fluorescence-activated cell sorting analysis. Purified CD34+ cells were subjected to ex vivo transmigration through double-coated transwell filter inserts, and an in vivo homing assay was performed in xenotransplanted NOD/SCID mice. RESULTS: UCB-derived CD34(bright) cells expressed significantly lower levels of CD49e, CD49f, and CXCR-4 than their mPB and BM counterparts. CD34+ cells from UCB (and BM) exhibited significantly lower ex vivo transmigration than those from mPB, which were largely blocked by neutralizing antibodies to CD49e or CD49f. Recombinant human tumor necrosis factor-alpha treatment enhanced ex vivo transmigration of CD34+ cells from UCB and BM by inducing expression of the matrix metalloproteinases MMP-2/MMP-9. Short-term treatment of UCB-derived CD34+ cells with rHu-stem cell factor (rHuSCF) up-regulated levels of the homing-related molecules with their increased ex vivo transmigratory and in vivo homing potential. CONCLUSION: Our results indicate that disadvantageous transmigratory behavior of HS/PCs from UCB, which might partly explain the delayed reconstitution after UCBT, can be reversed by ex vivo manipulation with rHuSCF.

Factors associated with outcomes of unrelated cord blood transplant: guidelines for donor choice.

OBJECTIVE: Optimizing cord blood donor selection based mainly on cell dose and human leukocyte antigen (HLA) disparities may further improve results of unrelated cord blood transplants (UCBT). MATERIALS AND RESULTS: We analyzed 550 UCBTs for hematologic malignancies reported to the Eurocord Registry. Main outcomes and prognostic factors were analyzed in univariable and multivariable analyses incorporating center and period effects and using death and relapse as competitive risks for nonfatal endpoints. Nucleated cell (NC) dose before freezing and number of HLA disparities had a significant influence on outcome. Cumulative incidence (CI) of neutrophil and platelet recovery was associated with the number of HLA mismatches, number of NC before freezing, and use of granulocyte colony-stimulating factor. Coexistence of HLA class I and II disparities and high CD34 cell dose in the graft were associated with graft-vs-host disease grades III-IV. CI of disease relapse was higher in matched transplants showing a graft-vs-leukemia effect increased in HLA-mismatched transplants. Overall 3-year survival was 34.4%. Prognostic factors for survival were recipient age, gender, and disease status. CONCLUSION: Our results provide indications for a better choice of cord blood units according to cord blood cell content and HLA.

Protective effects of thrombopoietin and stem cell factor on X-irradiated CD34+ megakaryocytic progenitor cells from human placental and umbilical cord blood.

In previous studies we characterized the radiosensitivity of CFU-megakaryocytes from human placental and umbilical cord blood and the effects of various early-acting cytokines. We found that the maximal clonal growth of CFU-megakaryocytes in vitro and maximal protection against X-ray damage were supported by a combination of thrombopoietin and stem cell factor. However, the mechanism by which the two cytokines exert a synergistic effect remained unclear, so we extended these studies to investigate the radioprotective action of synergistic thrombopoietin and stem cell factor on the survival of X-irradiated CD34(+) CFU-megakaryocytes. A combination of thrombopoietin and stem cell factor led to activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and to suppression of caspase 3 in X-irradiated CD34(+) cells. When PD98059 and various synthetic substrates-specific inhibitors of these proteins-were used, the combination had less effect on the clonal growth of X-irradiated CD34(+) CFU-megakaryocytes. However, the addition of wortmannin, a specific inhibitor of the phosphatidylinositol-3 kinase pathway, did not alter the synergistic action of thrombopoietin plus stem cell factor. We suggest that part of this synergistic effect can be explained by activation of mitogen-activated protein kinase and extracellular signal-regulated protein kinase and by suppression of the caspase cascade.

Effects of the combination of thrombopoietin with cytokines on the survival of X-irradiated CD34(+) megakaryocytic progenitor cells from normal human peripheral blood.

Combinations of thrombopoietin and cytokines that act on megakaryocyte development (stem cell factor, IL3, IL6, IL11, flt3 ligand (now known as FLT3LG), erythropoietin, GM-CSF and G-CSF were evaluated for their ability to enhance clonal growth in vitro of X-irradiated CD34(+) megakaryocytic progenitor cells (CFU-megakaryocytes) purified from normal human peripheral blood. These data were compared with corresponding results described previously for CD34(+) CFU-megakaryocytes from human placental/umbilical cord blood (I. Kashiwakura, Radiat. Res. 153, 144-152, 2000). All cytokines, except IL3, promoted thrombopoietin-induced colony formation, but they resulted in exponential radiation survival curves. No significant differences in the D(0) (46-61 cGy) and extrapolation number n (1.00-1.04) were observed between thrombopoietin alone and in combination with these cytokines. IL3 did not promote colony formation, but marked shoulders were observed on the survival curves (D(0) = 91 cGy, n = 2.83). Flow cytometric analysis of cells harvested from cultures of X-irradiated cells stimulated with thrombopoietin plus IL3 showed no significant differences in the expression of surface antigens and DNA ploidy distribution of megakaryocytes from the control. These findings suggest that IL3 plays a key role in promoting the survival of X-irradiated CD34(+) CFU-megakaryocytes from peripheral blood as well as those from cord blood, though the former are more radiosensitive.

Effects of osteogenic induction on mesenchymal cells from fetal and maternal parts of human placenta.

To clarify whether the mesenchymal cells derived from human placenta were available for bone regeneration, we investigated the effects of osteogenic induction on mesenchymal cells of fetal and maternal parts of the placenta. The osteogenic-induced mineralization in both types of cells was measured by von Kossa staining, and the calcium concentration and the expression of osteogenic markers were assayed by RT-PCR. In the mesenchymal cells of both parts, osteopontin, osteocalcin, alkaline phosphatase, and collagen type I, which are osteogenic markers, were expressed. Moreover, the mesenchymal cells of the fetal part of the placenta were mineralized for 3 weeks, but those of the maternal part were not. These results showed that the mesenchymal cells derived from human placenta had an osteogenic phenotype and that only the mesenchymal cells of the fetal part were capable of being used as a cell source for bone reconstitution.

Severe immune dysfunction after lethal neutron irradiation in a JCO nuclear facility accident victim.

The optimal treatment for the hematological toxicity of acute radiation syndrome (ARS) is not fully established, especially in cases of high-dose nonuniform irradiation by mixed neutrons and gamma-rays, because estimation of the irradiation dose (dosimetry) and prediction of autologous hematological recovery are complicated. For the treatment of ARS, we performed HLA-DRB1-mismatched unrelated umbilical cord blood transplantation (CBT) for a nuclear accident victim who received 8 to 10 GyEq mixed neutron and gamma-ray irradiation at the JCO Co. Ltd. nuclear processing facility in Tokaimura, Japan. Donor/ recipient mixed chimerism was attained; thereafter rapid autologous hematopoietic recovery was achieved in concordance with the termination of immunosuppressants. Immune function examined in vitro showed recovery of the autologous immune system was severely impaired. Although the naive T-cell fraction and the helper T-cell subtype 1 fraction were increased, the mitogenic responses of T-cells and the allogeneic mixed leukocyte reaction were severely suppressed. Endogenous immunoglobulin production was also suppressed until 120 days after the accident. Although skin transplantation for ARS was successful, the patient died of infectious complications and subsequent acute respiratory distress syndrome 210 days after the accident. These results suggest that fast neutrons in doses higher than 8 to 10 Gy cause complete abrogation of the human immune system, which may lead to fatal outcome even if autologous hematopoiesis recovers. The roles of transplantation, autologous hematopoietic recovery, chimerism, immune suppression, and immune function are discussed.

Effects of amifostine on the proliferation and differentiation of megakaryocytic progenitor cells.

This study investigated the effects of amifostine, a clinically usable radioprotector or chemoprotector, on the proliferation and differentiation of normal and X-irradiated cluster of differentiation 34 positive (CD34+) megakaryocytic progenitor cells (colony-forming unit in megakaryocytes, CFU-Meg) from human placental and umbilical cord blood (CB) in vitro. Amifostine significantly accelerated megakaryocyte colony formation in a plasma clot culture supplemented with recombinant human thrombopoietin because of an increase in immature CFU-Meg-derived large megakaryocyte colony formation. An analysis of the cells that were harvested from the culture showed that amifostine induced a 70- and an 83-fold increase in the total cell and CFU-Meg numbers, respectively, and produced hyperploid megakaryocytes of more than 8 N ploidy. The radioprotective effect of amifostine on the clonal growth of X-irradiated CD34+ CFU-Meg was observed by treatment before or after irradiation. These findings suggest that the action of amifostine extends from immature CFU-Meg to the terminal differentiation of megakaryopoiesis, and its radioprotective effect is shown in megakaryopoiesis and thrombopoiesis.