Showing papers in "International Journal of Hematology in 2002"

Iron Toxicity and Chelation Therapy

Abstract: Iron is an essential mineral for normal cellular physiology, but an excess can result in cell injury. Iron in low-molecular-weight forms may play a catalytic role in the initiation of free radical reactions. The resulting oxyradicals have the potential to damage cellular lipids, nucleic acids, proteins, and carbohydrates; the result is wide-ranging impairment in cellular function and integrity. The rate of free radical production must overwhelm the cytoprotective defenses of cells before injury occurs. There is substantial evidence that iron overload in experimental animals can result in oxidative damage to lipids in vivo, once the concentration of iron exceeds a threshold level. In the liver, this lipid peroxidation is associated with impairment of membrane-dependent functions of mitochondria and lysosomes. Iron overload impairs hepatic mitochondrial respiration primarily through a decrease in cytochrome C oxidase activity, and hepatocellular calcium homeostasis may be compromised through damage to mitochondrial and microsomal calcium sequestration. DNA has also been reported to be a target of iron-induced damage, and this may have consequences in regard to malignant transformation. Mitochondrial respiratory enzymes and plasma membrane enzymes such as sodium-potassium-adenosine triphosphatase (Na++K+-ATPase) may be key targets of damage by non-transferrin-bound iron in cardiac myocytes. Levels of some antioxidants are decreased during iron overload, a finding suggestive of ongoing oxidative stress. Reduced cellular levels of ATP, lysosomal fragility, impaired cellular calcium homeostasis, and damage to DNA all may contribute to cellular injury in iron overload. Evidence is accumulating that free-radical production is increased in patients with iron overload. Iron-loaded patients have elevated plasma levels of thiobarbituric acid reactants and increased hepatic levels of aldehyde-protein adducts, indicating lipid peroxidation. Hepatic DNA of iron-loaded patients shows evidence of damage, including mutations of the tumor suppressor gene p53. Although phlebotomy therapy is effective in removing excess iron in hereditary hemochromatosis, chelation therapy is required in the treatment of many patients who have combined secondary and transfusional iron overload due to disorders in erythropoiesis. In patients with β-thalassemia who undergo regular transfusions, deferoxamine treatment has been shown to be effective in preventing iron-induced tissue injury and in prolonging life expectancy. The use of the oral chelator deferiprone remains controversial, and work is continuing on the development of new orally effective iron chelators.

Functional Heterogeneity of Colony-Stimulating Factor : Induced Human Monocyte-Derived Macrophages

Abstract: Macrophages have various functions and play a critical role in host defense and the maintenance of homeostasis. However, macrophages are heterogeneous and exhibit a wide range of phenotypes with regard to their morphology, cell surface antigen expression, and function. When blood monocytes are cultured in medium alone in vitro, monocytes die, and colony-stimulating factors (CSFs) such as macrophage (M)-CSF or granulocyte-macrophage (GM)-CSF are necessary for their survival and differentiation into macrophages. However, M-CSF-induced monocyte-derived macrophages (M-MΦ) and GM-CSF-induced monocyte-derived macrophages (GM-MΦ) are distinct in their morphology, cell surface antigen expression, and functions, including Fcγ receptor mediated-phagocytosis, H2O2 production, H2O2 sensitivity, catalase activity, susceptibility to human immunodeficiency virus type 1 andMycobacterium tuberculosis, and suppressor activity. The characteristics of GM-MΦ resemble those of human alveolar macrophages.

Clinical and pathological criteria for the diagnosis of essential thrombocythemia, polycythemia vera, and idiopathic myelofibrosis (agnogenic myeloid metaplasia).

Abstract: A set of clinical and pathological criteria for the diagnosis and staging of Philadelphia chromosome-negative myeloproliferative disorders (Ph1--MPDs) is presented by including bone marrow histopathology as a significant tool to identify the early, manifest, and advanced stages of essential thrombocythemia (ET), polycythemia vera (PV), and idiopathic myelofibrosis/agnogenic myeloid metaplasia (IMF/AMM). This combined approach provides a pathognomonic clue to each of the different subtypes of Ph1--MPDs and further enables recognition of the various steps in the evolution of the myeloproliferative process. Increase and clustering of giant to large megakaryocytes with mature cytoplasm and multilobulated staghorn-like nuclei in a normal or only slightly increased cellular bone marrow represent major hallmarks of ET. Loose assemblies of small to giant pleiomorphic mega-karyocytes containing deeply lobulated nuclei together with a proliferation of erythro- and granulopoiesis (panmyelosis) are the specific lesions of PV. The initial prefibrotic and the overt and more advanced myelofibrotic stages of IMF/AMM show a pronounced proliferation of an abnormal megakaryo- and granulopoiesis dominated by clustered atypical medium-sized to giant megakaryocytes with cloud-like, bulbous, and often hyperchromatic nuclei, which are not seen in allied subtypes of MPDs including chronic myeloid leukemia (Ph1+-CML) and myelodysplastic syndromes (MDS). The presented clinical and pathological criteria modify the Polycythemia Vera Study Group (PVSG) proposals for the Ph1--MPDs by including bone marrow histopathology and are in keeping with features outlined in the new World Health Organization classification.The latter allows the differentiation of true ET from reactive thrombocytosis and from thrombocythemias as an eventually presenting finding in PV, IMF/AMM, MDS, and Ph1+-CML. Moreover, these diagnostic guidelines are able to separate latent and early PV from secondary erythrocytosis and to detect the prefibrotic and early stages of IMF/AMM. Myelofibrosis is not a feature of ET and is rarely observed in PV at time of diagnosis, but it becomes apparent during long-term follow-up and constitutes a prominent lesion during the course of IMF/ AMM. Life expectancy is almost normal in ET and is also not significantly altered during the first, but compromised during the second, decade of follow-up in PV. On the other hand, survival is substantially shortened in IMF/AMM, even for patients with throm-bocythemia as a frequent finding of prefibrotic and early stage IMF/AMM.

Human T lymphotropic virus type-I and adult T-cell leukemia in Japan.

Abstract: HTLV-I is the first retrovirus to be associated directly with human malignancy. In ATL-endemic areas, the rate of HTLV-I carriers is high. Both HTLV-I and ATL have been shown to be endemic in some regions of the world, especially in southwest Japan, the Caribbean islands, South Americas, and parts of Central Africa. Antibodies against HTLV-I have been found in over one million individuals, and more than 700 cases of ATL have been diagnosed each year in Japan alone. The cumulative incidence of ATL among HTLV-I carriers in Japan is estimated at 2.5% (3-5% in males, 1-2% in females). In endemic areas, HTLV-I Ab were found in the sera of 6 to 37 percent of healthy adults over 40 years of age. This clustering is thought to be due to the limited transmission of virus between socially isolated populations. The diagnostic criteria for HTLV-I associated ATL have been defined as follows. 1) Histologically and/or cytologically proven lymphoid malignancy with T cell antigens. 2) Abnormal T-lymphocytes present in the peripheral blood, except in the lymphoma type. 3) Serum specimens for all patients with ATL have HTLV-I Ab. 4) Demonstration of clonality of HTLV-I proviral DNA is a definite diagnosis of ATL. ATL shows diverse clinical features but can be divided into four subtypes: acute, chronic, smoldering, and lymphoma type. The pattern of HTLV-I transmission is through one of three different modes. Infected mothers can transmit the virus to newborns mainly via breast milk. The virus also can be transmitted from male to female by sexual intercourse, and through blood transfusion. Chemotherapy is not effective; the acute and lymphoma types have a poor prognosis. ATL is generally treated with curative intent using combination chemotherapy, although long-term success has been very limited. Unfortunately that advance did not translate into an improvement in the overall survival; the median remain 10 months. In contrast, smoldering ATL, or some cases of chronic ATL, may have a more protracted natural course, which may be compromised by aggressive chemotherapy. Alternative strategies for both acute and chronic forms are clearly needed. After infection of HTLV-I, there is a long latent period before onset of ATL. Analyses by PCR showed that clearly proliferation occurred in intermediate state or even carriers with high virus load. Such clonal proliferation might be preleukemic stage, which suggested that carriers with high virus load should be risk group to have ATL.

Pathogen inactivation of blood components: current status and introduction of an approach using riboflavin as a photosensitizer.

Abstract: Riboflavin is a naturally occurring compound and an essential human nutrient. Studies in the 1960s and 70s showed that it could be effective, when exposed to visible or UV light, in inactivating viruses and bacteria. This suggested to us that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products, because of its nucleic acid specificity and its limited tendency toward indiscriminate oxidation. The riboflavin molecule is a planar, conjugated ring structure with a sugar side chain that confers water solubility. The planar portion is capable of intercalating between the bases of DNA or RNA. Light activated riboflavin oxidizes guanine in nucleic acids, preventing replication of the pathogen's genome. Gambro BCT is developing processes using riboflavin and light to inactivate pathogens in plasma, platelet, and red cell products. We call these Pathogen Eradication Technology (PET) processes. Riboflavin is non-toxic; it must be present in the body for good health. The photo-byproducts formed in the PET processes are lumichrome and protein adducts. The photodegradation of riboflavin in the body is clearly shown by the decrease in its concentration in neonates who are treated with intense visible light to break down circulating bilirubin, which their immature livers cannot yet handle. A definitive lookback study showed no difference in cancer rates between the 55,000 children receiving this therapy in Denmark from 1977 through 1989 and nonirradiated controls. Gambro BCT is developing specific riboflavin-based PET processes for platelet concentrates, fresh frozen plasma, and packed red blood cells. In each, the process is being optimized to achieve high levels of inactivation of specific pathogens, while maintaining acceptable levels of product quality and activity. Extra- and intracellular HIV, BVDV (a model for HCV), and pseudorabies virus (a herpes virus) have been used to guide process development and validation. We have demonstrated 4 to 7 log10 reductions in the titers of these viruses, when they are spiked into blood products and irradiated in the presence of riboflavin. Porcine parvovirus, a tight-capsid, nonenveloped virus is more resistant, a finding in all experimental inactivation approaches. A range of bacteria implicated in platelet and red cell transfusion injuries and deaths, including S. aureus, E. coli, K. pneumoniae, and Y. enterocolitica, are being used to validate antibacterial efficacy. The PET platelet process involves the addition of riboflavin to platelets in plasma, illumination of the product, storage of the product and transfusion without further manipulation. The lack of toxicity of the treatment byproducts permits this ease of use. Quality of the platelets throughout storage has been assessed by pH, PO2, lactate, hypotonic shock response, morphology, glucose, and GMP-140 expression. In vitro function is well maintained. The levels seen are within the range of those reported in commonly transfused products. Radiolabeled transfusion studies of treated platelets have been carried out in primates to determine a preliminary measure of their in-vivo circulation. The in vivo recoveries and survivals of treated and control platelets did not differ. This work suggests that an endogenous photosensitizer, riboflavin, which has an extremely good safety profile, can inactivate high levels of a broad range of viruses and bacteria in platelet concentrates, fresh frozen plasma, and in red blood cells, preserving the activity and functionality of the components. Planned animal and clinical studies are expected to solidify this suggestion into a well-characterized process which can be safely and readily applied to reduce the risks of transfusion transmitted disease.

Molecular and Cellular Biology of von Willebrand Factor

Abstract: von Willebrand factor (VWF) is a plasma protein that performs 2 main functions in hemostasis: it mediates platelet adhesion to the injured vessel wall, and it carries and protects coagulation factor VIII. VWF is synthesized through a multistep process in endothelial cells and megakaryocytes as a very large polymer composed of identical disulfide-linked 250-kd subunits. In endothelial cells, VWF not only directs the formation of its own storage granules, the Weibel-Palade bodies, but it also acts as a chaperone molecule to direct other proteins, such as P-selectin, into these granules. Upon stimulation of the endothelium, the Weibel-Palade bodies will be translocated to the plasma membrane, and their contents will be secreted into the plasma milieu. The expression of VWF can be regulated at different levels by a number of genetic and environmental factors, resulting in control of its activity. New roles for VWF, especially in inflammatory processes, have recently been suggested, indicating that some aspects of this well-studied protein remain to be investigated.

Nationwide Study of Idiopathic Thrombocytopenic Purpura in Pregnant Women and the Clinical Influence on Neonates

Abstract: Idiopathic thrombocytopenic purpura (ITP) occurs more commonly in young women during the reproductive years. To obtain information for management of ITP in pregnancy, we performed a nationwide retrospective survey. Findings from a total of 284 pregnant women with ITP and their 286 newborn infants were available for analysis. The bleeding tendency at delivery was managed chiefly with corticosteroid, intravenous high-dose γulin, and platelet transfusion. Maternal complications occurred in 77 cases (27.1%) and were frequently seen in cases with poor control of ITP. Neonatal abnormalities, which were not influenced by the clinical state of the mother, occurred at a frequency of 17.8%. Thrombocytopenia in neonates occurred in 48 cases (22.4%), and bleeding tendency was found in 16 cases (6.3%) without severe bleeding. Prediction of thrombocytopenia in neonates was difficult. However, infants from splenectomized mothers with well-controlled ITP showed thrombocytopenia more frequently than those from nonsplenectomized mothers. Mothers treated with steroids at doses greater than 15 mg/day showed a high frequency of maternal complications and fetal abnormal body weight. These observations will be useful in the management of pregnant women with ITP and their infants.

The international bone marrow transplant registry.

Abstract: The first successful transplants of hematopoietic stem cells were done in 1968 in three children with congenital immune deficiency diseases. In each instance, stem cells were collected from the bone marrow of sibling donors who were genotypically HLA identical or closely HLA matched to the recipient. Since then, thousands of patients have received hematopoietic stem cell transplants to treat malignant and non-malignant diseases. Current estimates of annual numbers of stem cell transplants are about 50,000 worldwide, with growth at a rate of 10-15% per year. The reasons for increasing use include proven and potential efficacy in many diseases, better understanding of the appropriate timing of transplantation and patient selection, greater availability of donors and better techniques for determining HLA match, greater ease of stem cell collection, and improved supportive care resulting in less transplant-related morbidity and mortality. About two-thirds of hematopoietic stem cell transplants use autologous cells, generally collected from peripheral blood by leukapheresis. The remainder are allogeneic transplants, most commonly from HLA-identical sibling donors, and most often using cells collected directly from bone marrow.

von Willebrand Factor—Cleaving Protease and Upshaw-Schulman Syndrome

Abstract: Vascular endothelial cell (EC)-produced plasma von Willebrand factor (vWF) plays a critical role in primary hemostasis through its action of anchoring platelets onto the injured denuded subendothelial matrices under high shear stress. Unusually large vWF multimers (UL-vWFMs), present in plasma immediately after release from ECs, are most biologically active, but they are soon cleaved and degraded into smaller vWFMs by a specific plasma protease, termed vWF-cleaving protease (vWF-CPase), in normal circulation. Recent studies on the relationship between UL-vWFMs and vWF-CPase, together with its autoantibody (inhibitor) have brought about a clear discrimination between thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Furthermore, a congenital deficiency of this enzyme activity has been shown to cause Upshaw-Schulman syndrome, a complex constitutional bleeding diathesis. Successful purification of vWF-CPase revealed that this enzyme is composed of a single polypeptide with a molecular mass of approximately 190 kd, and its complementary DNA cloning unambiguously indicated that it is uniquely produced in the liver and its gene is located on chromosome 9q34. The messenger RNA of vWF-CPase had a span of 4.6 kb, and its enzyme was designated ADAMTS 13. The predicted complete amino acid sequence of this enzyme consisted of 1427 residues, including a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat (TSP1), a cysteine-rich domain, an ADAMTS spacer, 7 additional TSP1 repeats, and 2 CUB domains.

Biological significance of proliferation, apoptosis, cytokines, and monocyte/macrophage cells in bone marrow biopsies of 145 patients with myelodysplastic syndrome

Abstract: Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-α was correlated with TGF-β (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-β level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model, TGF-β emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-α, TGF-β also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.

Genetic abnormalities of Bernard-Soulier syndrome.

Abstract: Bernard-Soulier Syndrome (BSS) is an autosomal recessive bleeding disorder due to quantitative or qualitative abnormalities in the glycoprotein (GP) Ib/IX/V complex, the platelet receptor for von Willebrand factor. BSS is characterized by giant platelets, thrombocytopenia, and prolonged bleeding time, and the hallmark of this disorder is the absence of ristocetininduced platelet agglutination. In the last 10 years, the molecular and genetic bases of many GPIb/IX/V defects have been elucidated, providing a better understanding of primary hemostasis and structure-function relations of the complex. Thus far, more than 30 mutations of the GPIbα, GPIbβ, or GPIX genes have been described in BSS. Recent studies also have shown that the phenotypes caused by mutations in the subunits of the GPIb/IX/V span a wide spectrum, from the normal phenotype, to isolated giant platelet disorders/macrothrombocytopenia, to full-blown BSS and platelet-type von Willebrand disease. Although recent progress in molecular biology has clarified the genotype-phenotype relationships of the GPIb/IX/V disorders, a close examination of platelet morphology on blood smears is still indispensable for a proper diagnosis. In this review, we summarize recent advances in the molecular basis of BSS with special emphasis on giant platelets and the genetic characteristics of Japanese BSS.Int J Hematol. 2002; 76: 319-327.

A comparative double-blind randomized trial of activated protein C and unfractionated heparin in the treatment of disseminated intravascular coagulation.

Abstract: A randomized prospective double-blind trial was performed to compare the safety and efficacy of human activated protein C (APC) and unfractionated heparin for the treatment of disseminated intravascular coagulation (DIC). One hundred thirty-two patients with DIC were enrolled in this study: 63 patients received APC (12.5 U [2.5 μg]/kg body wt per hour) and 69 patients received heparin (8 U/kg body wt per hour) by intravenous infusion for 6 days. Forty-nine APC-treated patients and 55 heparintreated patients were evaluated for efficacy, and 52 APC-treated patients and 55 heparin-treated patients were evaluated for safety. The 2 groups were similar with respect to sex, age, body weight, underlying diseases, and coagulation/fibrinolysis parameters before treatment. Aggravation of bleeding was seen after treatment in 8 patients receiving heparin, but in none of the patients receiving APC. The number of patients who showed alleviation of bleeding was significantly higher in the APC group than the heparin group (P = .009). The effects on DIC-related organ dysfunction were not significantly different between the 2 groups. Fibrinogen-fibrin degradation products, D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PIC) were all significantly decreased by treatment in both groups. Fibrinogen, protein C, and antithrombin were significantly increased in the APC group, whereas only protein C was significantly increased in the heparin group. Platelet count in the nonleukemic group was significantly increased in those patients receiving APC but not increased in those patients receiving heparin. Improvement of coagulation/ fibrinolysis was assessed by scoring 4 parameters (soluble fibrin monomers, D-dimer, TAT, and PIC), and the results indicated that the APC group showed significantly greater improvement than the heparin group (P = .046). There was, however, no significant difference in the rate of complete recovery from DIC between the 2 groups. The rate of death from any cause within 28 days after treatment was 20.4% in the APC group, significantly lower than the 40% death rate observed in the heparin group (P < .05). There were no severe adverse events in either group. These results suggest that APC in a relatively small dosage can improve DIC more efficiently than can heparin, without increasing bleeding, and may be a better alternative.

The role of the macrophage in apoptosis: hunter, gatherer, and regulator.

Abstract: Clearance of cellular corpses is a critical feature of apoptosis in vivo during development, tissue homeostasis, and resolution of inflammation. As the professional phagocytes of the body, macrophages play a key role in this process. By recognizing emerging signals using several different receptors, macrophages engulf apoptotic cells swiftly and efficiently. In addition, the binding of apoptotic cells profoundly down-regulates the ability of the macrophage to produce inflammatory mediators by inducing the release of antiinflammatory mediators. Finally, macrophages may actually induce cell death in specific cells during embryogenesis.Abnormalities of apoptotic cell clearance may contribute to the pathogenesis of chronic inflammatory diseases, including those of autoimmune etiology. It is also possible that certain malignant tumor cells co-opt the mechanisms for apoptotic cell clearance to avoid immune surveillance by subverting macrophage and dendritic cell responses.

Macrophage-specific gene expression: current paradigms and future challenges.

Abstract: Cells of the mononuclear phagocyte lineage include macrophages, microglia, osteoclasts, and myeloid dendritic cells. These cell types are all derived from blood monocytes, which are the product of hematopoietic stem cell differentiation. In this review we use specific examples of macrophage-expressed genes to illustrate potential regulatory strategies for directing macrophage-specific gene expression. The examples we have chosen-the human c-fes gene, the murine spi-1 (PU.1) gene, the human RANTES promoter, and the human CD68 gene-illustrate different aspects of constitutive and inducible gene expression in macrophages. One important challenge for future work in this field will be to identify the molecular events that dictate lineage decisions during the differentiation of mononuclear phagocytes from hematopoietic progenitor cells. Another important goal will be to understand how groups of macrophage genes are coordinately expressed in response to physiological, immunological, and inflammatory stimuli. A better understanding of macrophage gene expression may find application in gene therapy, genetic vaccination, and the development of new antiinflammatory drugs.

Gene therapy of X-linked severe combined immunodeficiency.

Abstract: Severe combined immunodeficiency (SCID) conditions appear to be the best possible candidates for a gene therapy approach. Transgene expression by lymphocyte precursors should confer to these cells a selective growth advantage that gives rise to long-lived T-lymphocytes. This rationale was used as a basis for a clinical trial of the SCID-X1 disorder caused by common γ (γc) gene mutations. This trial consists of ex vivo retroviral-mediated (MFG-B2 γc vector) γc gene transfer into marrow CD34+ cells in CH-296 fibronectin fragment—coated bags. Up to now, 9 patients with typical SCID-X1 diagnosed within the first year of life and lacking an HLA-identical donor have been enrolled. More than 2 years’ assessment of 5 patients and more than 1 year for 7 patients provide evidence for full development of functional, mature T-cells in the absence of any adverse effects. Functional transduced natural killer cells are also detectable, although in low numbers. All but 1 patient with T-cell immunity have also developed immunoglobulin production, which has alleviated the need for intravenous immunoglobulin substitution despite a low detection frequency of transduced B-cells. These 8 patients are doing well and living in a normal environment. This yet successful gene therapy demonstrates that in a setting where transgene expression provides a selective advantage, a clinical benefit can be expected.Int J Hematol. 2002;76:295-298.

The Notch Pathway: Modulation of Cell Fate Decisions in Hematopoiesis

Abstract: The hematopoietic system is maintained by a rare population of hematopoietic stem cells (HSC) that are thought to undergo self-renewal as well as continuously produce progeny that differentiate into the various hematopoietic lineages. However, the mechanisms regulating cell fate choices by HSC and their progeny have not been understood. Results of most studies support a stochastic model of cell fate determination in which growth factors support only the survival or proliferation of the progeny specified along a particular lineage. In other developmental systems, however, Notch signaling has been shown to play a central role in regulating fate decisions of numerous types of precursors, often inhibiting a particular (default) pathway while permitting self-renewal or differentiation along an alternative pathway. There is also accumulating evidence that the Notch pathway affects survival, proliferation, and cell fate choices at various stages of hematopoietic cell development, including the decisions of HSC to self-renew or differentiate and of common lymphoid precursors to undergo T- or B-cell differentiation. These data suggest that the Notch pathway plays a fundamental role in the development and maintenance of the hematopoietic system.

Long-term feto-maternal microchimerism: nature's hidden clue for alternative donor hematopoietic cell transplantation?

Abstract: During pregnancy, fetal hematopoietic cells carrying paternal human leukocyte antigens (HLA) migrate into maternal circulation, and, vice versa, maternal nucleated cells can be detected in fetal organs and umbilical cord blood, indicating the presence of bidirectional cell traffic between mother and fetus. By taking advantage of fluorescence in-situ hybridization or polymerase chain reaction-based techniques, researchers recently found that postpartum persistence of such reciprocal chimerism was common among healthy individuals and may sometimes cause tissue chimerism.Although the biological significance of long-lasting feto-maternal microchimerism is unknown, a number of investigations have suggested its association with the development of “autoimmune” diseases such as systemic sclerosis. However, the very common presence of feto-maternal microchimerism among subjects without any autoimmune attack may allow us the more appealing hypothesis that it is an indicator for the acquired immunological hyporesponsiveness to noninherited maternal or fetal HLA antigens. An offspring’s tolerance to noninherited maternal antigens has been clinically suggested by the retrospective analysis of renal transplantations or haploidentical hematopoietic stem cell transplantations, and whether postpartum mothers can tolerate paternally derived fetal antigens is an intriguing question. Although an exact linkage between microchimerism and transplantation tolerance is yet to be elucidated, long-term acceptance of a recipient’s cell in the donor may have a favorable effect on preventing the development of severe graft-versus-host disease, and the donor cell microchimerism in the recipient might facilitate the graft acceptance. If this concept holds true, HLA-mismatched hematopoietic stem cell transplantation would be more feasible among haploidentical family members mutually linked with feto-maternal microchimerism. Further studies are warranted to investigate the potential role of feto-maternal microchimerism in human transplantation medicine.

The role of WT1 in oncogenesis: tumor suppressor or oncogene?

Abstract: Although originally identified as a tumor suppressor gene, WT1 is overexpressed in a variety of hematologic malignancies and solid tumors, including acute leukemia, breast cancer, malignant mesothelioma, renal cell carcinoma, and others. Overex-pression of both wild-type and mutant WT1 has been reported. In some cases, this finding represents overexpression of a gene that should be expressed at lower levels, but in other cases, WT1 is expressed at high levels in a tissue type in which there is normally no expression at all. In this review, the mechanisms of altered WT1 expression are explored, including changes in promoter methylation. WT1 target genes that may be important for oncogenesis are discussed, as is the use of WT1 expression as a diagnostic tool. The prognostic implications of altered WT1 expression and the potential for immunotherapy aimed at WT1 are also discussed.

Expression of the antiapoptosis gene survivin in human leukemia.

Abstract: Loss of the inhibition of apoptosis is important in leukemogenesis and may influence the prognosis. Survivin is an inhibitor of apoptosis that shows selective expression during fetal development and in human malignancies. Survivin expression was examined in human leukemias using the reverse transcriptase-polymerase chain reaction. Survivin gene expression was detected in 17 of 31 patients with acute myelocytic leukemia and 11 of 16 patients with acute lymphocytic leukemia but was not identified in normal bone marrow cells. Survivin expression was lower in patients with M3 acute myelocytic leukemia than in patients with other types of acute leukemia. Survivin was not detected in the chronic phase of chronic myelocytic leukemia but was observed in 5 of 7 patients with chronic myelocytic leukemia in blastic crisis. These findings suggest a relationship between survivin gene expression and hematopoietic cell differentiation. In fact, survivin gene expression was down-regulated during the differentiation of HL-60 cells after treatment with dimethyl sulfoxide or all-trans-retinoic acid. Moreover, the disease-free survival rates of patients with survivin expression were lower than in patients without survivin expression. Accordingly, survivin may have a role in leukemogenesis as well as in other malignancies. Detecting survivin may also provide prognostic information.

New insights into role of microenvironment in multiple myeloma.

Abstract: Multiple Myeloma (MM) is a malignant disease of terminally differentiated B cells. It most likely originates in a B cell which has traversed the germinal center and has been exposed there extensively to antigens based on the high number of somatic mutations in the complementarity determining regions. The cell of origin is either a plasmablast, or more likely, a memory B-cell. Typically MM goes through different phases from indolent (MGUS, smoldering myeloma) to overt myeloma and then to a fulminant phase, characterized by extramedullary manifestations, high LDH, immature morphology and increased proliferation rate. In the indolent phase, the disease already has acquired major cytogenetic abnormalities as demonstrated by FISH and DNA flow cytometry. It has a gene pattern very similar to myeloma cells on gene array analysis. In the early stages of overt MM, the myeloma cells are completely dependent upon the micro-environment for their growth and survival. The interaction between myeloma cells and micro-environment causes bone disease, genetic instability and more importantly, drug-resistance, which is caused by upregulation of anti-apoptotic factors, resistance to apoptosis induced by FAS and TRAIL activation, and by cell adhesion-induced growth arrest. In this phase of the disease, MM is susceptible to chemotherapy, if delivered with adequate intensity. In the fulminant phase of MM, myeloma cells have acquired sufficient genetic alternations to become completely independent of the micro-environment which allows them to grow at extramedullary sites. Because of the many DNA breaks necessary for immature B cells to become mature plasma cells, B cells already have inherent genetic instability. DNA breaks are necessary for VDJ recombinations, somatic mutations and isotype switching and it is therefore not surprising that genetic alternations frequently occur at the Ig heavy chain site at 14q32, which is abnormal in three quarters of myeloma patients. Some of the translocations with 14q32 involve terminal fragments of chromosomes and can not be diagnosed with standard cytogenetics. Cytogenetic abnormalities are found in 30–35% of newly diagnosed patients and require sufficient proliferation of MM cells to find enough analyzable mitoses. The cytogenetic abnormalities are typically complex, involving ≥3 chromosomes in 80% of patients. Almost all chromosomes can be involved in deletions, additions or translocations of genetic material. Our group has repeatedly stressed the prognostic significance of chromosome 13 deletion by conventional cytogenetics. The role of chromosome 13 deletion by FISH is less clear. In addition to chromosome 13 deletion, the presence of a hypodiploid or hypotetraploid karyotye also carries a poor prognosis. Frequently, deletions of chromosome 13 and hypodiploidy go hand in hand. It remains unclear what specific gene confers the poor prognosis to patients with deletion 13. The issues of bone disease, drug resistance and cytogenetics will be addressed in detail during this presentation.

The relationship of aplastic anemia and PNH

Abstract: Bone marrow failure has been regarded as one of the triad of clinical manifestations of paroxysmal nocturnal hemoglobinuria (PNH), and PNH in turn has been described as a late clonal disease evolving in patients recovering from aplastic anemia. Better understanding of the pathophysiology of both diseases and improved tests for cell surface glycosylphosphatidylinositol (GPI)-linked proteins has radically altered this view. Flow cytometry of granulocytes shows evidence of an expanded PNH clone in a large proportion of marrow failure patients at the time of presentation: in our large NIH series, about 1/3 of over 200 aplastic anemia cases and almost 20% of more than 100 myelodysplasia cases. Clonal PNH expansion (rather than bone marrow failure) is strongly linked to the histocompatability antigen HLA.-DR2 in all clinical varieties of the disease, suggesting an immune component to its pathophysiology. An extrinsic mechanism of clonal expansion is also more consistent with knock-out mouse models and culture experiments with primary cells and cell lines, which have failed to demonstrate an intrinsic proliferative advantage for PNH cells. DNA chip analysis of multiple paired normal and PIG-A mutant cell lines and lymphoblastoid cells do not show any consistent differences in levels of gene expression. In aplastic anemia/PNH there is surprisingly limited utilization of the V-beta chain of the T cell receptor, and patients’ dominant T cell clones, which are functionally inhibitory of autologous hematopoiesis, use identical CDR3 regions for antigen binding. Phenotypically normal cells from PNH patients proliferate more poorly in culture than do the same patient’s PNH cells, and the normal cells are damaged as a result of apoptosis and overexpress Fas. Differences in protein degradation might play a dual role in pathophysiology, as GPI-linked proteins lacking an anchor would be predicted to be processed by the proteasome machinery and displayed in a class I H.A. context, in contrast to the normal pathway of cell surface membrane recycling, lysosomal degradation, and presentation by class II HLA. The strong relationship between a chronic, organ-specific immune destructive process and the expansion of a single mutant stem cell clone remains frustratingly enigmatic but likely to be the result of interesting biologic processes, with mechanisms that potentially cna be extended to the role of inflammation in producing premalignant syndromes.

Stem cell repair in ischemic heart disease: an experimental model.

Abstract: Bone marrow stem cells (BMSC) from adult mice are now believed to generate non-hematopoietic cell types. This newly defined property is referred to as stem cell plasticity. We tested the potential of lineage negative c-kit positive (Lin- c-kit+), GFP+ BMSC to differentiate into cardiac myocytes in myocardial infarcts produced by ligation of the left coronary artery. At 9 days post-transplant the hearts showed a band of developing GFP+ myocytes within the damaged myocardium. These GFP+ myocytes were positive for cardiac specific myosin and early expressed transcription factors. Endothelial cells and smooth muscle cells also developed from the donor bone marrow cells. Left ventricular end diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP) were improved. Lin-c- kit- cells did not regenerate myocardium. We next tested the ability of cytokine-mobilized BMSC to regenerate myocardium. Nuclei in regenerating cardiomyocytes were positive for Csx/Nkx 2.5, GATA-4 and MEF2. Cytoplasmic proteins included desmin, nestin and connexin 43. Regenerating arterioles consisted of endothelial cells and smooth muscle cells positive for Ki67, and flkl. These regenerating vessels contained circulating TER119 positive red blood cells. Repair of infarcted myocardium resulted in improved heart function and survival. At day 27 after cytokine treatment and surgery, 11 of 15 mice survived compared with 9 of 52 non-treated mice. Left ventricular ejection fraction in infarcted hearts in cytokine-treated mice was 48%, 62% and 114% higher than the ejection fraction in non-treated mice at 9, 16 and 26 days following coronary artery occlusion. These findings demonstrate that circulating autologous stem cells traffic to the ischemic, infarcted myocardium and undergo differentiation into cardiomyocytes and vascular structures. We conclude that adult BMSC have the potential for repair in acute, ischemic heart disease.

Chronic graft-versus-host disease and late effects after hematopoietic stem cell transplantation

Abstract: Late effects following HSCT are related to either the transplant process or to the transplant preparative regimen. Problems related to the transplant process include delayed recovery of the immune system and chronic GVHD. Chronic GVHD presents between 3–14 months post-HSCT in approximately 20% of matched sibling transplants and 40% of matched unrelated donor recipients. Most commonly involved sites are skin, mouth, liver, gastrointestinal tract, and eye. Patients with platelet count <100,000/ml and receiving cortocosteroid therapy at day 80 with any clinical manifestations of chronic GVHD require prolonged immune suppressive therapy with prednisone, cyclosporine ±other agents. Treatment should be administered until all clinical and pathological signs and symptoms of chronic GVHD have resolved which may take one to several years. Problems related to the transplant preparative regimen include those involving the endocrine system, eyes, lungs, bone, and development of secondary malignancies. Endocrine deficiencies include growth failure with growth hormone (GH) deficiency, overt hypothyroidism, primary gonadal failure, Type 1 or Type 2 diabetes, and exocrine pancreatic insufficiency. These problems develop at any time post-HSCT, but usually occur within the first few years and should be treated with appropriate hormone supplementation. Eye problems are primarily related to development of cateracts secondary to total body irradiation (TBI) or prolonged corticosteroid use. Cateracts developing after fractionated frequently do not require removal. Pulmonary problems may be due to bronchiolitis obliterans (BO) or to restrictive lung disease. BO may be associated with chronic GVHD and may respond to chronic GVHD therapy. Restrictive lung disease does not occur for many years after HSCT. There is not therapy for this problem. Development of decreased bone mineral density (BMD) is related to GH deficiency and/or corticosteroid therapy. Treatment includes withdrawal of corticosteroids, administration of GH and calcium, Vitamin D and antiresorptive agents. All malignant disease survivors are at risk for development of secondary malignancies including survivors of HSCT. Recipients of TBI are at highest risk as are children. All pediatric and adult survivors of HSCT should be followed for their life-time for development of delayed effects of transplantation.

Management of bleeding disorders by prohemostatic therapy.

Abstract: Pro-hemostatic therapy aims at an improvement of hemostasis, which may be achieved by amelioration of primary hemostasis, stimulation of fibrin formation or inhibition of fibrinolysis. These treatment strategies may be applied to specifically correct a defect in one of the pathways of coagulation, but have in some situations also been shown to be effective in reducing bleeding in patients without a primary defect in coagulation. Besides the transfusion of platelets in case of thrombocytopenia or severe platelet disorders, a pharmacological improvement of primary hemostasis may be achieved by the administration of desmopressin. The administration of DDAVP results in a marked increase in the plasma concentration of Von Willebrand factor (and associated coagulation factor VIII) and (also by yet unexplained additional mechanisms) a remarkable potentiation of primary hemostasis as a consequence. DDAVP is used for the prevention and treatment of bleeding in patients with von Willebrand disease or mild hemophilia A, and further in patients with an impaired function of primary hemostasis, such as in patients with uremia, liver cirrhosis or in patients with aspirin-associated bleeding. Based on the current insight that activation of coagulation in vivo predominantly proceeds by the tissue factor/factor VII(a) pathway, recombinant factor VIIa has been developed as a prohemostatic agent and has recently become available for clinical use. Indeed, in uncontrolled clinical studies this compound has been shown to exert a potent procoagulant activity and appeared to be highly effective in the prevention and treatment of bleeding, although most experience so far has been obtained in patients with severe and complicated coagulation defects. At present, a more general use of this agent for bleeding patients without an apparent coagulation defect is the subject of a number of ongoing clinical trials. Agents that exert anti-fibrinolytic activity are aprotinin and the group of lysine analogues. The pro-hemostatic effect of these agents proceeds not only by the inhibition of fibrinolysis (thereby shifting the procoagulant/anticoagulant balance towards a more procoagulant state), but also due to a protective effect on platelets, as has been demonstrated at least for aprotinin. The mechanism of this platelet-protective effect has, besides a potential prevention of plasmin-mediated loss of platelet receptors not been elucidated. Whether the pro-hemostatic effect of the anti-fibrinolytic agents will eventually result in a higher incidence of thromboembolic complications is still a matter of debate (see further), however, this has so far not been shown in straightforward clinical trials.

Analyzing the roles of mast cells and basophils in host defense and other biological responses

Abstract: The sudden and systemic activation of mediator release from mast cells and basophils that can occur when some sensitized subjects are challenged by minute amounts of specific antigen (eg, from an insect sting or peanuts) can result in fatal ana-phylaxis, a reaction that arguably represents the most grotesque imbalance between the cost and benefit of an immune response. Why then do mast cells and basophils continue to exist and, in the case of mast cells, populate almost all vascularized tissues? This review will consider the roles of mast cells and basophils in health and disease, emphasizing particularly their proven or potential functions in host defense. We will also describe briefly some approaches to investigate mast cell and basophil functions in vivo, including the use of mast cells generated directly from embryonic stem cells in vitro.

Resveratrol, a natural product derived from grapes, is a new inducer of differentiation in human myeloid leukemias.

Abstract: A natural product, resveratrol (3,4,40-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other food products, is known as a cancer chemopreventive agent. We studied the in vitro biological activity of this compound by examining its effect on proliferation and differentiation in myeloid leukemia cell lines (HL-60, NB4, U937,THP-1, ML-1, Kasumi-1) and fresh samples from 17 patients with acute myeloid leukemia. Resveratrol (20 μM, 4 days) alone inhibited the growth in liquid culture of each of the 6 cell lines. Resveratrol (10 μM) enhanced the expression of adhesion molecules (CD11a, CD11b, CD18, CD54) in each of the cell lines except for Kasumi-1. Moreover, resveratrol (25 μM, 4 days) induced 37% of U937 cells to produce superoxide as measured by the ability to reduce nitroblue tetrazolium (NBT). The combination of resveratrol (10 μM) and all-trans-retinoic acid (ATRA) (50 nM, 4 days) induced 95% of the NB4 cells to become NBT-positive, whereas <1% and 12% of the cells became positive for NBT after a similar exposure to either resveratrol or ATRA alone, respectively. In U937 cells exposed to resveratrol (25 μM, 3 days), the binding activity of nuclear factor-κB (NFκB) protein was suppressed. Eight of 19 samples of fresh acute leukemia cells reduced NBT after exposure to resveratrol (20 μM, 4 days). Taken together, these findings show that resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells.

Molecular Pathogenesis of Fanconi Anemia

Abstract: Fanconi anemia (FA) is a rare autosomal recessive chromosomal breakage disorder characterized by the childhood onset of aplastic anemia, developmental defects, cancer susceptibility, and cellular hypersensitivity to DNA—cross-linking agents FA patients can be divided into at least 8 complementation groups (FA-A, FA-B, FA-C, FA-D1, FA-D2, FA-E, FA-F, and FA-G) FA proteins encoded by 6 cloned FA genes (FANCA,FANCC,FANCD2,FANCE,FANCF, andFANCG) cooperate in a common pathway, culminating in the monoubiquitination of FANCD2 protein and colocalization of FANCD2 and BRCA1 proteins in nuclear foci These BRCA1 foci have been implicated in the process of homologous recombination-mediated DNA repair In this review, we will summarize the current progress in the field of FA research and highlight some of the potential functions of the FA pathway in DNA-damage response

Thalassemia and abnormal hemoglobin.

Abstract: Thalassemia and abnormal hemoglobins are common genetic disorders in Asia. Thalassemia is not only an important public health problem but also a socio-economic problem of many countries in the region. The approach to deal with the thalassemic problem is to prevent and control birth of new cases. This requires an accurate identification of the couple at high risk for thalassemia. However, the diagnosis of thalassemia carrier states need several tests which are not practical for screening the population at large. Recently we have used two simple laboratory tests to screen for potential thalassemia carriers and hemoglobin E individuals. There is also a new development in using the automatic HPLC to diagnose thalassemic diseases and the carriers. This system gives both qualitative and quantitative analysis of hemoglobin components in the same run with good precision and reproducibility. The system has been applied to study thalassemia and abnormal Hb in adult and cord blood. This system has enabled us to do both prenatal and postnatal diagnosis of thalassemia within the few minutes. However, none of these screening tests can accurately give specific diagnosis of the thalassemia genotype. Specific thalassemia mutation can be carried out by DNA analysis. Many DNA techniques have been used for point mutation detection and small deletion. For the last few years there is a development of DNA chip technology that has been applied for thalassemia mutation as well. Clinically, thalassemia is very heterogeneous in the manifestation. In spite of seemingly identical genotypes, severity of.beta thalassemic patients can vary greatly. Heterogeneity in the clinical manifestation of beta thalassemic diseases may occur from the nature of beta globin gene mutation, alpha thalassemia gene interaction and difference in the amount of Hb F production which is partly associated with a specific beta globin haplotype. However, there is still some beta thalassemia cases that have a mild clinical symptom without those known genetic fators interaction suggesting that there are other additional factors responsible for the mildness of the disease.

Transferrin receptor in tissue and serum: updated clinical significance of soluble receptor

Abstract: The transferrin receptor is an essential component of cellular uptake of iron, and it binds to serum transferrin. Recently, 2 different types of transferrin receptors have been recognized: transferrin receptor (TfR or transferrin receptor 1) and transferrin receptor 2. Most cells possess a ubiquitous system controlling the biosynthesis of TfR at the posttranscriptional level to avoid excess iron influx into the cells through TfR. During the process of recycling of transferrin receptors, some are shed and appear as soluble or serum transferrin receptors. Measurement of serum transferrin receptor is a new marker of iron metabolism that reflects body iron stores and total erythropoiesis. It has been shown that serum transferrin receptor to ferritin ratios have significant predictive value for differentiating iron deficiency anemia from noniron deficiency anemia, such as anemia of chronic disorders, whereas serum ferritin is the only significant independent predictor of iron deficiency anemia.

Transcriptional Regulation of Myelopoiesis

Abstract: A common myeloid progenitor gives rise to both granulocytes and monocytes. The early stages of granulopoiesis are mediated by the C/EBPalpha, PU.1, RAR, CBF, and c-Myb transcription factors, and the later stages require C/EBPepsilon, PU.1, and CDP. Monocyte development requires PU.1 and interferon consensus sequence binding protein and can be induced by Maf-B, c-Jun, or Egr-1. Cytokine receptor signals modulate transcription factor activities but do not determine cell fates. Several mechanisms orchestrate the myeloid developmental program, including cooperative gene regulation, protein:protein interactions, regulation of factor levels, and induction of cell cycle arrest.