Ernest Beutler, MD and Carol West
Background: Gaucher disease results from the accumulation of glucosylceramide (glucocerebroside) in tissues of affected persons. Patients sharing the same genotype present with widely varying degrees of lipid storage and of clinical manifestations.
Objectives: To determine whether variation in the glucosylceramide synthase (UDPGlucose ceramide glucosyltransferase) gene, which encodes the enzyme that regulates the synthesis of glucocerebroside, could account for the variability and clinical manifestations.
Methods: Patients homozygous for the 1226G (N370S) mutation, the most common in the Ashkenazi Jewish population, were investigated. The exons and flanking sequences of the gene were sequenced using DNA derived from five very mild Gaucher disease patients and four patients with relatively severe Gaucher disease. Results: One polymorphism was found in the coding region, but this did not change any amino acids. Seven other polymorphisms were found in introns and in the 5' untranslated region. Some of these were single nucleotide polymorphisms; others were insertions. The mutations appear to be in linkage equilibrium and none were found with a significantly higher frequency in either severe or mildly affected individuals.
Conclusions: Mutations in the glucosylceramide synthase gene do not appear to count for the variability in expression of the common Jewish Gaucher disease mutation.
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.
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.
HSPG = heparan sulfate proteoglycan
GBM = glomerular basement membrane
Job Harenberg, MD, Jorg Ingrid, MD and Fenyvesi Tivadar, MD
Background: Venous thromboembolic diseases are treated initially with low molecular weight heparin followed by oral coumarins.
Objectives: To investigate an orally available direct thrombin inhibitor for the acute treatment of venous thromboembolism as well as for prophylaxis of recurrent events.
Methods: The direct thrombin inhibitor ximelagatran was compared with subcutaneous LMW heparins followed by oral warfarin in a double-blind randomized prospective multicenter trial in patients with acute VTE. A pharmacokinetic study was performed in the VTE patients. For assessing the prevention of recurrent VTE, double-blind prospective randomized studies were conducted as follows: a) ximelagatran compared to warfarin for 6 months, and b) prolonged anticoagulation of ximelagatran vs. placebo for 18 months after termination of 6 months coumarin therapy.
Results: Two dose-finding studies and the pharmacokinetic analysis of ximelagatran in acute VTE were completed. About 2,500 patients were randomized to investigate 2 x 36 mg ximelagatran versus 2 x 1 mg/kg body weight enoxaparin followed by warfarin. The study hypothesized that the efficacy was equal in both treatment regimens for recurrent VTE documented by objective methods. The second study, with 1,234 patients, aimed to demonstrate a reduced incidence of recurrent thromboembolic events documented by objective methods after 18 months of treatment with 2 x 24 mg ximelagatran daily compared to placebo.
Conclusion: These large-scale clinical trials will soon yield the results of the comparison between oral ximelagatran and subcutaneous LMW heparin for treatment of acute VTE, and of warfarin for prophylaxis of recurrent events for 6 months and for a prolonged prophylaxis for another 18 months.
LMW = low molecular weight
VTE = venous thromboembolism
David G. Motto, MD, PhD, James A. Williams, MD and Laurence A. Boxer, MD
Background: Chronic childhood autoimmune hemolytic anemia is an uncommon disorder that is associated with significant morbidity. Treatment with high dose steroids, splenectomy and frequent blood transfusions results in a myriad of complications including growth failure, bone demineralization, Cushing’s syndrome, immunosuppression, and transfusional hemosiderosis.
Objectives: To investigate the efficacy of the monoclonal anti-CD20 antibody, rituximab, in treating children with AIHA.
Methods: Four children with chronic AIHA, including two with prior splenectomy, who were dependent on high dose steroids and refractory to other immunosuppressive regimens were treated with four to six weekly doses of rituximab at a dose of 375 mg/m2.
Results: All four patients became transfusion-independent and were taken off prednisone completely. Adverse effects included infusion-related reactions that were mild, and infectious complications of Pneumocystis carinii pneumonia and varicella pneumonia.
Conclusions: Treatment with rituximab appears promising for refractory AIHA; it may obviate the need for prednisone and may result in sustained disease remissions in some patients.
Itzchak Beiran, MD, Tamar Talmon, MD and Benjamin Miller, MD
Background: The prevalence of traumatic hyphema as well as the distribution of its severity varies between different patient populations. Treatment recommendations in the literature differ significantly among various published reports. This lack of a uniformly accepted treatment probably reflects the different characteristics of this pathology among the populations investigated and calls for a population-adjusted treatment recommendation.
Objectives: To report the characteristics and functional outcome of patients with traumatic hyphema and to discuss possible recommendations regarding the use of ε‑aminocaproic acid.
Methods: A prospective, non-randomized study was conducted among 154 consecutive patients with traumatic hyphema, including data collection of ophthalmic status at various time points, the presence or absence of secondary hemorrhage, and final visual acuity.
Results: Of the 154 eyes studied over 3½ years, nearly 90% had hyphema of grade 1 or less, 3 (3.25%) experienced rebleeding, and 2 (1.3%) – neither of which rebled – needed surgical intervention. None of the four patients who experienced final visual acuity of 6/40 or less suffered rebleeding.
Conclusion: The use of ε‑aminocaproic acid in the studied population was unjustified and routine use of e-aminocaproic acid in our patient population is probably not indicated. A treatment policy regarding e-aminocaproic acid use should be adjusted to the population being treated.
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.
G-CSF = granulocyte colony-stimulating factor
MDS = myelodysplastic syndrome
AML = acute myeloid leukemia
Shifra Sela, PhD, Revital Shurtz-Swirski, PhD, Jamal Awad, MD, Galina Shapiro, MSc, Lubna Nasser, MSc, Shaul M. Shasha, MD and Batya Kristal, MD
Background: Cigarette smoking is a well-known risk factor for the development of endothelial dysfunction and the progression of atherosclerosis. Oxidative stress and inflammation have recently been implicated in endothelial dysfunction.
Objectives: To assess the concomitant contribution of polymorphonuclear leukocytes to systemic oxidative stress and inflammation in cigarette smokers.
Methods: The study group comprised 41 chronic cigarette-smoking, otherwise healthy males aged 45.0 ± 11.5 (range 31–67 years) and 41 male non-smokers aged 42.6 ± 11.3 (range 31–65) who served as the control group. The potential generation of oxidative stress was assessed by measuring the rate of superoxide release from separated, phorbol 12-myristate 13-acetate-stimulated PMNL and by plasma levels of reduced (GSH) and oxidized (GSSG) glutathione. Inflammation was estimated indirectly by: a) determining the in vitro survival of PMNL, reflecting cell necrosis; b) in vivo peripheral PMNL counts, reflecting cell recruitment; and c) plasma alkaline phosphatase levels, indicating PMNL activation and degranulation.
Results: PMA-stimulated PMNL from cigarette smokers released superoxide at a faster rate than PMNL from the controls. Smokers had decreased plasma GSH and elevated GSSG levels. In vitro incubation of control and smokers' PMNL in sera of smokers caused necrosis, while control sera improved smoker PMNL survival. Smokers' PMNL counts, although in the normal range, were significantly higher than those of controls. Plasma ALP levels in smokers were significantly higher than in controls and correlated positively with superoxide release and PMNL counts.
Conclusions: Our study shows that PMNL in smokers are primed in vivo, contributing concomitantly to systemic oxidative stress and inflammation that predispose smokers to endothelial dysfunction, and explains in part the accelerated atherosclerosis found in smokers.
PMNL = polymorphonuclear leukocytes
PMA = phorbol 12-myristate 13-acetate
GSH = reduced glutathione
GSSG = oxidized glutathione
ALP = alkaline phosphatase