Jane Zhao, MD, Hsiao-Nan Hao, MD and William D. Lyman, PhD
Background: Experimental and clinical protocols are being developed for the cryopreservation of human hematopoietic progenitor cells. However, the effect of these procedures on the potential for HPC to repopulate bone marrow is unknown.
Objectives: To examine the effect of cryopreservation on the ability of fetal human liver HPC, which include CD34+ cells and long-term culture-initiating cells, to repopulate immunodeficient non-obese diabetic/severe combined immunodeficiency mouse bone marrow.
Methods: Groups of sublethally irradiated NOD/SCID mice were injected intravenously with cryopreserved or freshly isolated fetal human liver HPC.
Results: Seven weeks after transplantation, flow cytometric analysis of bone marrow samples showed that mice that received the transplanted cells (either cryopreserved or freshly isolated) demonstrated both lymphoid and myeloid differentiation as well as the retention of a significant fraction of CD34+ cells. Conclusions: Cryopreserved fetal human liver-derived HPC appear to be capable of initiating human cell engraftment in NOD/SCID mouse bone marrow and open the possibility of using cryopreserved fetal human liver HPC for gene manipulation, gene transfusion therapy, and transplantation purposes.
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Jorge Rouvier, MD, Claudio Gonzalez, MD, Alejandra Scazziota, PhD and Raul Altman, MD
Background: Elevated fibrinogen, considered an independent risk factor for coronary disease, stratifies an individual as high risk for coronary disease. A risk marker requires little intra-individual variability during a long period.
Objectives: To establish intra-individual variability of fibrinogen levels in patients with coronary disease.
Methods: We investigated fibrinogen levels prospectively in four blood samples drawn from 267 patients with a history of arterial disease (arterial group) and from 264 patients with cardiac valve replacements (valvular group). The samples were taken during the course of 78.7 and 78.8 days from the arterial and valvular groups respectively.
Results: Marked intra-individual dispersion with a reliability coefficient of 0.541 was found in the arterial group and 0.547 in the valvular group. The Bland-Altman test showed low probability to obtain similar results in different samples from the same individual. These results show large intra-individual variability, with similarities in the arterial as well as in the valvular group.
Conclusions: It is not possible to stratify a patient by a specific fibrinogen dosage.
Avi Katz, MD, David J. Van-Dijk, MD, Helena Aingorn, PhD, Arie Erman, MD, Malcolm Davies, MD, David Darmon, MD, Hagit Hurvitz, MD and Israel Vlodavsky, PhD
Background: Decreased heparan sulfate proteoglycan content of the glomerular basement membrane has been described in proteinuric patients with diabetic nephropathy. Heparanase is an endo-b-D-glucuronidase that cleaves negatively charged heparan sulfate side chains in the basement membrane and extracellular matrix.
Objectives: To investigate whether urine from type I diabetic patients differs in heparanase activity from control subjects and whether resident glomerular cells could be the source of urinary heparanase.
Methods: Using soluble 35S-HSPG and sulfate-labeled extracellular matrix we assessed heparanase activity in human glomerular epithelial cells, rat mesangial cells, and urine from 73 type I diabetic patients. Heparanase activity resulted in the conversion of a high molecular weight sulfate-labeled HSPG into heparan sulfate degradation fragments as determined by gel filtration analysis.
Results: High heparanase activity was found in lysates of both epithelial and mesangial cells. Immunohistochemical staining localized the heparanase protein to both glomeruli capillaries and tubular epithelium. Heparanase activity was detected in the urine of 16% and 25% of the normoalbuminuric and microalbuminuric diabetic patients, respectively. Urine from 40 healthy individuals did not posses detectable heparanase. Urinary heparanase activity was associated with worse glycemic control.
Conclusion: We suggest that heparanase enzyme participates in the turnover of glomerular HSPG. Hyperglycemia enhances heparanase activity and/or secretion in some diabetic patients, resulting in the loss of albumin permselective properties of the GBM.
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HSPG = heparan sulfate proteoglycan
GBM = glomerular basement membrane
by Melvin H. Freedman, MD, FAAP, FRCPC and Blanche P. Alter, MD, FAAP, MPH
Background: Granulocyte colony-stimulating factor has had a major impact on the management of severe chronic neutropenia – a collective term referring to congenital, idiopathic, or cyclic neutropenia. Almost all patients respond to G-CSF with increased neutrophils, reduced infections, and improved survival. Some responders with congenital neutropenia (termed Kostmann’s syndrome herein) and Shwachman-Diamond syndrome have developed myelodysplastic syndrome and acute myeloid leukemia, which raises the question of the role of G-CSF in pathogenesis. The issue is complicated because both disorders have a propensity for MDS or AML as part of their natural history.
Objective and Methods: To address this, the Severe Chronic Neutropenia International Registry used its large database of chronic neutropenia patients treated with G-CSF to determine the incidence of malignant myeloid transformation in the two disorders, and its relationship to treatment and to other patient characteristics.
Results: As of January 2001, of the 383 patients with congenital forms of neutropenia in the Registry, 48 had MDS or AML (crude rate, about 12.5%). No statistically significant relationships were found between age at onset of MDS or AML and patient gender, G-CSF dose, or duration of G-CSF therapy. What was observed, however, was the multistep acquisition of aberrant cellular genetic changes in marrow cells from Kostmann’s syndrome patients who transformed, including activating ras oncogene mutations, clonal cytogenetic abnormalities, and G-CSF receptor mutations. The latter in murine models produces a hyperproliferative response to G-CSF, confers resistance to apoptosis, and enhances cell survival.
Conclusions: Since Kostmann’s syndrome and Shwachman-Diamond syndrome are inherited forms of bone marrow failure, G-CSF may accelerate the propensity for MDS/AML in the genetically altered stem and progenitor cells, especially in those with G-CSF receptor and ras mutations (82% and 50% of Kostmann’s syndrome patients who transform, respectively). Alternatively, and equally plausible, G-CSF may simply be an innocent bystander that corrects neutropenia, prolongs patient survival, and allows time for the malignant predisposition to declare itself. Only careful long-term follow-up of the cohort of patients receiving G-CSF will provide the answer.
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G-CSF = granulocyte colony-stimulating factor
MDS = myelodysplastic syndrome
AML = acute myeloid leukemia
Arnon Blum, MD, Julia Sheiman, MD and Yonathan Hasin, MD
Jacob Cohen, MSc, Lia Supino-Rosin, MSc, Eran Barzilay, BSc, Ronit Eisen-Lev, DMD, Moshe Mittelman, MD and Drorit Neumann, PhD
Ami Neuberger, MD, Sigal Fishman, MD and Ahuva Golik, MD