Aberrant regulation of superantigen responses during T-cell reconstitution and graft-versus-host disease in immunodeficient mice

Acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation is associated with impaired deletion and anergy of host-reactive T cells. To elucidate the immunoregulatory events that may contribute to such dysregulated T-cell responses in GVHD, we studied superantigen (SAg) responses after adoptive T-cell transfer into severe combined immunodeficient (SCID) mice. SAg responses are normally regulated by mechanisms involving deletion and anergy, with SAg-reactive T cells typically being deleted rapidly in vivo. In a SCID mouse model of GVHD, however, allogeneic host SAg-reactive T cells were not deleted rapidly, but rather persisted in increased numbers for several months. Moreover, depending on the timing of SAg stimulation and the numbers of T cells transferred, dysregulation (impaired deletion and anergy) of SAg responses could be demonstrated following the adoptive transfer of syngeneic T cells into SCID mice as well. Transgenic T-cell receptor-bearing KJ1-26.1(+) T cells were then used to determine the fate of weakly reactive T cells after adoptive transfer and SAg stimulation. When transferred alone, KJ1-26.1(+) T cells demonstrated impaired deletion and anergy. In the presence of more strongly staphylococcal enterotoxin B (SEB)-reactive T cells, however, KJ1-26.1(+) T cells were regulated normally, in a manner that could be prevented by inhibiting the effects of more strongly SEB-reactive cells or by increasing the level of activation of the KJ1-26.1(+) T cells themselves. We suggest that the control mechanisms that normally regulate strongly activated T cells in immunocompetent animals are lost following adoptive transfer into immunodeficient hosts, and that this impairment contributes to the development of GVHD.     

Pyruvate kinase-catalyzed ATP-formation in human red blood cell membranes

Summary Previous studies on the linkage between enzymatically catalyzed ATP-generating reactions in the red blood cell membrane and the sodium and potassium transport in the control of overall glycolysis of human erythrocytes were controversial. In this study a significant amount of pyruvate kinase activity is shown to be localized within the membrane. Membrane fragments produce 20.5mol of ATP per 10¹⁰ membranes per hour from phosphoenolpyruvate and ADP. The kinetics of the membrane-localized pyruvate kinase do not differ from those of the enzyme from hemolysates. The results clearly document the presence of the second ATP-generating enzyme of glycolysis, pyruvate kinase, in human red blood cell membranes. The main fraction of the enzyme is deeply hidden in the lipid layers of the membrane. It can be demasked by mechanical desintegration of membranes at high levels of activity. It is suggested that the amount of the membrane-localized fraction of pyruvate kinase is related to the clinical severity of the hemolytic process in pyruvate kinase deficiency.Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Schr 86/13     

Systems biology of the human red blood cell

While the potential possibilities of systems biology appear to hold great promise, there are still many challenges to overcome, notably lack of complete characterization of biological components and their interactions and ways to measure these changes experimentally. The comparative simplicity of the human red cell sidesteps much of these challenges. Detailed models of human red cell metabolism have been developed for the last quarter century and continued development of these models has resulted in the ability to make functional phenotype predictions, in silico. Additionally, existing experimental proteomic and metabolomic technologies now provide the capability to perform individualized characterization of erythrocytes. While an experimentally testable systems biology model of most human cells is presently out of reach, it may be achievable with the red cell.     

Ineffective erythropoiesis with reduced red blood cell survival in serotonin-deficient mice

Serotonin (5-HT) has long been recognized as a neurotransmitter in the central nervous system, where it modulates a variety of behavioral functions. Availability of 5-HT depends on the expression of the enzyme tryptophan hydroxylase (TPH), and the recent discovery of a dual system for 5-HT synthesis in the brain (TPH2) and periphery (TPH1) has renewed interest in studying the potential functions played by 5-HT in nonnervous tissues. Moreover, characterization of the TPH1 knockout mouse model (TPH1(-/-)) led to the identification of unsuspected roles for peripheral 5-HT, revealing the importance of this monoamine in regulating key physiological functions outside the brain. Here, we present in vivo data showing that mice deficient in peripheral 5-HT display morphological and cellular features of ineffective erythropoiesis. The central event occurs in the bone marrow where the absence of 5-HT hampers progression of erythroid precursors expressing 5-HT(2A) and 5-HT(2B) receptors toward terminal differentiation. In addition, red blood cells from 5-HT-deficient mice are more sensitive to macrophage phagocytosis and have a shortened in vivo half-life. The combination of these two defects causes TPH1(-/-) animals to develop a phenotype of macrocytic anemia. Direct evidence for a 5-HT effect on erythroid precursors is provided by supplementation of the culture medium with 5-HT that increases the proliferative capacity of both 5-HT-deficient and normal cells. Our thorough analysis of TPH1(-/-) mice provides a unique model of morphological and functional aberrations of erythropoiesis and identifies 5-HT as a key factor for red blood cell production and survival.     

Immune reconstitution after blood cell transplantation

Immune clinical events and pattern of recovery of total lymphocytes, T cells and CD4+/CD8+ T cell subsets, B cells and NK cells, were analysed after allogeneic blood cell transplantation (BCT) for 12 months following transplant (n = 33). Results were compared with immune recovery after allogeneic bone marrow transplantation (BMT) (n = 15) and autologous blood cell transplantation (n = 19). Rates of acute GVHD were comparable in the two allotransplantation groups. Fourteen out of 17 evaluable alloBCT group patients have presented an extensive cGVHD. Total T cell, CD4+T cells and CD56+NK cell reconstitution were improved in alloBCT group vs alloBMT group. CD4+/CD8+ ratio evolution were improved in alloBCT group vs alloBMT and autoBCT groups. These results confirm that rapid immune recovery is associated with allogeneic blood stem cell transplantation.     

The human red blood cell glyoxalase system in diabetes mellitus

Methylglyoxal and other alpha-oxoaldehydes are formed from glycolytic intermediates and may be involved in the development of diabetic microangiopathy. Glyoxalase I and glyoxalase II metabolise methylglyoxal to D-lactic acid, via the intermediate S-D-lactoylglutathione. The activities of the glyoxalase enzymes and the concentrations of methylglyoxal and S-D-lactoylglutathione were measured in erythrocytes of 45 control and 85 diabetic subjects (41 with retinopathy and 44 uncomplicated). The concentration of S-D-lactoylglutathione was increased in diabetic patients vs. controls (21.4 +/- 9.3 vs. 12.4 +/- 4.8 mumol/l, P less than 0.001), as was methylglyoxal (3.6 +/- 2.3 vs. 1.4 +/- 0.2 mumol/l, P less than 0.001). There were no significant differences in the activities of glyoxalase I and glyoxalase II between diabetic patients and controls. For insulin-dependent patients only, those without retinopathy had a higher activity of glyoxalase II than those with retinopathy (P less than 0.05). A group of age- and duration-matched insulin-dependent diabetic patients with retinopathy also had a higher activity of glyoxalase I compared with a group of diabetic patients without retinopathy (P less than 0.025). This study provides evidence for elevated concentrations of oxoaldehydes in diabetes mellitus which might have pathogenic significance.     

Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice

Unseparated or Ficoll-Hypaque (Pharmacia, Piscataway, NJ)--fractionated human cord blood cells were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice. High levels of multilineage engraftment, including myeloid and lymphoid lineages, were obtained with 80% of the donor samples as assessed by DNA analysis, fluorescence-activated cell sorting (FACS), and morphology. In contrast to previous and concurrent studies with adult human bone marrow (BM), treatment with human cytokines was not required to establish high-level human cell engraftment, suggesting that neonatal cells either respond differently to the murine microenvironment or they provide their own cytokines in a paracrine fashion. Committed and multipotential myelo- erythroid progenitors were detected using in vitro colony assays and FACS analysis of the murine BM showed the presence of immature CD34+ cells. In addition, human hematopoiesis was maintained for at least 14 weeks providing further evidence that immature hematopoietic precursors had engrafted the murine BM. This in vivo model for human cord blood- derived hematopoiesis will be useful to gain new insights into the biology of neonatal hematopoietic cells and to evaluate their role in gene therapy. There is growing evidence that there are ontogeny-related changes in immature human hematopoietic cells, and therefore, the animal models we have developed for adult and neonatal human hematopoiesis provide useful tools to evaluate these changes in vivo.     

Metabolic remodeling of the human red blood cell membrane

The remarkable deformability of the human red blood cell (RBC) results from the coupled dynamic response of the phospholipid bilayer and the spectrin molecular network. Here we present quantitative connections between spectrin morphology and membrane fluctuations of human RBCs by using dynamic full-field laser interferometry techniques. We present conclusive evidence that the presence of adenosine 5′-triphosphate (ATP) facilitates non-equilibrium dynamic fluctuations in the RBC membrane that are highly correlated with the biconcave shape of RBCs. Spatial analysis of the fluctuations reveals that these non-equilibrium membrane vibrations are enhanced at the scale of spectrin mesh size. Our results indicate that the dynamic remodeling of the coupled membranes powered by ATP results in non-equilibrium membrane fluctuations manifesting from both metabolic and thermal energies and also maintains the biconcave shape of RBCs.     

Subcutaneous and intrahepatic growth of human hepatoblastoma in immunodeficient mice

BACKGROUND/AIMS: Hepatoblastoma is the most frequent malignant pediatric liver tumor. Approximately 25% of hepatoblastoma patients cannot be cured with current treatment protocols. Additional treatment options must, therefore, be developed. Subcutaneous animal models for hepatoblastoma exist, but a more physiologic intrahepatic model is lacking. METHODS: The alpha-fetoprotein-expressing hepatoblastoma-cell lines HepT1, HuH6 and the childhood hepatocellular carcinoma-cell line HepG2 were injected subcutaneously and intrasplenically into NMRI nu/nu mice. Tumor growth was monitored by measuring tumor size for subcutaneous and serum human alpha-fetoprotein levels for intra-abdominal tumors. Tumors were characterized microscopically. RESULTS: Subcutaneous tumor growth occurred in 70% (7/10) of mice injected with HuH6 and 50% (5/10) of mice injected with HepG2. HepT1 did not form tumors. Accumulation of serum alpha-fetoprotein reflected tumor growth. Intrasplenic growth was seen in 50% (14/27, HuH6) and 10% (3/10, HepG2) of the mice, with only HuH6 forming intrahepatic tumors in 25% (7/27) of the mice. Growth pattern and alpha-fetoprotein production were similar at the subcutaneous and intra-abdominal location. Intrahepatic grafting occurred by metastatic spread from the spleen, produced well-defined nodules, and was accompanied by a weakened expression of the hepatocyte marker carbamoylphosphate synthetase, and the canalicular markers CD10 and cytokeratin7. The expression of cytokeratin18 and -19, active caspase3, and beta-catenin was increased. There were no lung metastases. CONCLUSIONS: We established an intrahepatic mouse model for human hepatoblastoma, in which tumor growth could be monitored by serum alpha-fetoprotein levels. Engrafting in the liver occurred by metastatic spread from the spleen and was accompanied by some loss of differentiation features.     

Preferential localisation of red cell vacuoles to pathologically shaped human red blood cells

The percentage of pitted erythrocytes and Howell-Jolly bodies in peripheral blood samples of 51 individuals following posttraumatic splenectomy and 20 patients splenectomized because of various haematological diseases differed significantly from each other (p less than 0.001) and from that of healthy controls (p less than 0.001). The percentage of pitted erythrocytes was significantly higher in pathologically shaped red blood cells (RBCs) (acanthocytes, schizocytes, elliptocytes) than in normal discoid shaped RBCs (p less than 0.001). As the number of pits per RBC showed great individual variations, a scoring system for the evaluation of pitted RBCs is proposed.     

Growth of primary human acute leukemia in severe combined immunodeficient mice

Seven populations of human leukaemic cells were implanted i.v. into sublethally irradiated severe combined immunodeficient (scid) mice. Growth of leukaemia was monitored by labelling murine peripheral blood (PB) cells with an anti-HLA monoclonal antibody and flow cytometric analysis. Two of the populations transplanted were fresh acute lymphoblastic leukaemia (ALL) bone marrow (BM) cells which both caused sustained proliferative growth in scid mice. Human cells accounted for up to a mean of 87% of the total nucleated cells (TNC) in the PB of these mice between weeks 12-15. One of these populations was passaged into fresh mice and frank leukaemia was again established. Three populations of cryopreserved acute myeloblastic leukaemia (AML) cells (2 obtained from PB and 1 from BM) and one population of cryopreserved biphenotypic acute leukaemia BM cells, only grew to a maximum of 4% within the 15 week period of the experiment. A cell population from an AML cell line (HL60), however, did engraft and proliferate resulting in a rapid deterioration of these mice between weeks 3-6 when the proportion of human cells accounted for 9% of the TNC in the PB.     

Characteristics of zinc binding to human red blood cell membranes

The objective of the present study was to standardize the analysis of zinc binding on human red blood cell (RBC) membranes in 20 normal adults. The displacement studies revealed that at the maximal stable zinc concentration tested (600 μM), 57% (mean) of the bound ⁸⁵Zn was displaced and to displace half maximal ⁶⁵Zn, the stable zinc concentration was 300 μM. Scatchard plots revealed two classes of binding sites for zinc on RBC membranes: one with higher affinity, Kd = 1.20 × 10^?5 M (site I), and the other with lower affinity, Kd = 2.77 × M (site II). Binding sites occupancy was 97% means and 58.5% means for sites I and II, respectively. The displacement was affected by temperature, membrane protein concentration, freezing, thawing, and dialysis. Other metal cations, including Co^+*, Fe⁺⁺, and Mn⁺⁺, had very little effect on ⁶⁵Zn displacement, in contrast copper displaced ⁶⁵Zn from its binding sites on RBC membranes. Zinc binding to RBC membranes was rapid and readily reversible in a dynamic equilibrium with its binding sites. It is anticipated that this method will be applicable to studies of a wide variety of diseases specifically related to zinc metabolism in humans as well as in animals. © 1994 Wiley-Liss, Inc.     

Purinergic signaling inhibits human acute myeloblastic leukemia cell proliferation, migration, and engraftment in immunodeficient mice

Extracellular ATP and UTP nucleotides increase the proliferation and engraftment potential of normal human hematopoietic stem cells via the engagement of purinergic receptors (P2Rs). In the present study, we show that ATP and UTP have strikingly opposite effects on human acute myeloblastic leukemia (AML) cells. Leukemic cells express P2Rs. ATP-stimulated leukemic cells, but not normal CD34+ cells, undergo down-regulation of genes involved in cell proliferation and migration, whereas cell-cycle inhibitors are up-regulated. Functionally, ATP induced the inhibition of proliferation and accumulation of AML cells, but not of normal cells, in the G0 phase of the cell cycle. Exposure to ATP or UTP inhibited AML-cell migration in vitro. In vivo, xenotransplantation experiments demonstrated that the homing and engraftment capacity of AML blasts and CD34+CD38- cells to immunodeficient mice BM was significantly inhibited by pretreatment with nucleotides. P2R-expression analysis and pharmacologic profiling suggested that the inhibition of proliferation by ATP was mediated by the down-regulation of the P2X7R, which is up-regulated on untreated blasts, whereas the inhibition of chemotaxis was mainly mediated via P2Y2R and P2Y4R subtypes. We conclude that, unlike normal cells, P2R signaling inhibits leukemic cells and therefore its pharmacologic modulation may represent a novel therapeutic strategy.     

Restoration of red blood cell volume following 2-unit red blood cell apheresis

BACKGROUND AND OBJECTIVES: Blood donors who weigh at least 130 lbs (59 kg) and have a haematocrit of at least 40 V per cent can donate 2 units of blood, from which a 360-ml volume of red blood cells (RBC) can be isolated. This study was carried out in seven healthy male blood donors to assess the restoration of the RBC volume 1 month following a 2-unit RBC apheresis procedure. MATERIALS AND METHODS: RBC volumes were measured prior to donation and 4 weeks after the 2-unit RBC apheresis procedure without oral iron supplementation. RESULTS: Four weeks after the removal of 2 units of RBC from the male donors not supplemented with oral iron, the RBC volume was restored to 92% of the precollection value. The 360-ml volume of RBC collected represented 12-19% of the donor's original RBC volume. CONCLUSIONS: Male donors can safely donate 2 units of RBC and will restore a mean of 92% of their RBC volume within 1 month without iron supplementation.     

细胞因子体外扩增脐血CD+34细胞移植SCID鼠人类免疫功能的建立

目的：探讨扩增的脐血ＣＤ３４细胞体内造血功能。方法：ＦＬＴ３ 配体联合血小板生成素体外培养脐血ＣＤ＋３４细胞２～３ 周后移植ＳＣＩＤ鼠,采用ＦＣＭ 检测ＳＣＩＤ鼠骨髓、外周血、脾脏中的人类ＣＤ４５、ＣＤ３、ＣＤ１９抗原。结果：脐血ＣＤ＋３４细胞扩增１０～３７ 倍,移植６．６×１０４～６．９×１０５ 脐血ＣＤ＋３４细胞于ＳＣＩＤ鼠,６ 周后１０ 只ＳＣＩＤ鼠骨髓中均检测到人类ＣＤ４５抗原（０．２％ ～４．０％ ）,６ 只ＳＣＩＤ鼠外周血、脾脏中检测到人类ＣＤ３ 抗原（０．１％ ～２．３％ ）,３ 只ＳＣＩＤ鼠中外周血、脾脏中检测到人类ＣＤ１９抗原（０．３％ ～１．３％ ）。结论：ＦＬＴ３ 配体联合血小板生成素体外扩增的脐血ＣＤ＋３４细胞能重建ＳＣＩＤ鼠人类免疫功能。     

Correction of X-linked immunodeficient mice by competitive reconstitution with limiting numbers of normal bone marrow cells.

Gene therapy for inherited disorders is more likely to succeed if gene-corrected cells have a proliferative or survival advantage compared with mutant cells. We used a competitive reconstitution model to evaluate the strength of the selective advantage that Btk normal cells have in Btk-deficient xid mice. Whereas 2,500 normal bone marrow cells when mixed with 497,500 xid cells restored serum IgM and IgG3 levels to near normal concentrations in 3 of 5 lethally irradiated mice, 25,000 normal cells mixed with 475,000 xid cells reliably restored serum IgM and IgG3 concentrations and the thymus-independent antibody response in all transplanted mice. Reconstitution was not dependent on lethal irradiation, because sublethally irradiated mice all had elevated serum IgM and IgG3 by 30 weeks postreconstitution when receiving 25,000 normal cells. Furthermore, the xid defect was corrected with as few as 10% of the splenic B cells expressing a normal Btk. When normal donor cells were sorted into B220(+)/CD19(+) committed B cells and B220(-)/CD19(-) cell populations, only the B220(-)/CD19(-) cells provided long-term B-cell reconstitution in sublethally irradiated mice. These findings suggest that even inefficient gene therapy may provide clinical benefit for patients with XLA.     

Failure of stem cell factor to ameliorate AZT-induced anemia in immunodeficient mice

Abstract: Recent attempts to reduce 3′azido-3′deoxythymidine (AZT)-induced anemia in AIDS patients have focused both on AZT dose reduction and on the use of recombinant cytokines. The newly cloned cytokine stem cell factor (SCF) is a potent regulator of hematopoietic progenitor cell proliferation. Therefore, we attempted to ameliorate AZT-induced anemia using stem cell factor (SCF) in the LP-BM5 murine leukemia virus-induced model of AIDS (MAIDS). SCF was administered with oral AZT for up to 1 month, and effects on erythropoiesis examined. SCF alone increased both splenic BFU-E and CFU-E. AZT alone also increased the number of splenic BFU-E and CFU-E. SCF, administered to AZT-treated MAIDS mice, did not further enhance these increases. SCF increased bone marrow cellularity in AZT-treated MAIDS mice. However, the total number of bone marrow BFU-E was unaffected. In contrast, AZT, SCF, and the combination significantly decreased bone marrow CFU-E. SCF alone increased the absolute numbers of peripheral blood reticulocytes in MAIDS mice, but did not increase reticulocyte numbers in AZT-treated mice. SCF did not significantly increase hematocrits in either control or AZT-treated mice. Further studies are needed to maximize the differentiating capacity of the enlarged erythroid progenitor cell pool induced by SCF.