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
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.
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
Bernard M. Babior, PhD
The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O2- (superoxide) at the expense of NADPH. The O2- then dismutes to H2O2, which serves to oxidize Cl- to HOCl, a potent microbicidal agent that is used by leukocytes to kill invading microorganisms. This oxidation is catalyzed by myeloperoxidase. O2 is also used to make other microbicidal oxidants, some in reactions with nitric oxide. The oxidase itself is highly complex, consisting of four unique subunits and Rac2. In the resting cell, two of the subunits, p22PHOX and gp91PHOX, are located in the membrane, and the other two, p47PHOX and p67PHOX, are in the cytosol. The electron-carrying components of the oxidase are
located in gp91PHOX; the NADPH binding site is generally regarded to be in gp91PHOX as well, but there is some evidence that it may be in p67PHOX. When the oxidase is activated, p47PHOX is phosphorylated at specific sites, and the cytosolic components plus Rac2 migrate to the membrane to assemble the active oxidase.
Peter C. Harpel, MD and Nasreen S. Haque, PhD
Chemokines and their receptors play regulatory roles in inflammatory reactions. Lipoprotein(a) is an atherogenic lipoprotein, however the mechanisms of its actions are not defined. Our interest in chemokines and their receptors was stimulated by the finding that incubation of Lp(a) with human umbilical vein endothelial cells produced a conditioned medium that was chemotactic for human monocytes. Since infiltration of monocytes into the vessel wall is an early lesion in atherosclerosis, this finding provided a novel mechanism to explain the relationship between Lp(a) and atherosclerosis. The chemoattractant produced by HUVEC was identified as CCL1/I-309, a CC chemokine previously reported to be secreted by stimulated monocytes/macrophages and T lymphocytes. CCR8, the CCL1 receptor, was identified on endothelial cells, and CCL1 was found to be a chemoattractant for these cells. Most recently we demonstrated functional CCR8 on human vascular smooth muscle cells and found that the Lp(a)-HUVEC conditioned medium is a chemoattractant for these cells. CCL1 increased metalloproteinase-2 production by HUVEC, an activity that enables these cells to remodel the vascular matrix. These studies suggest that CCR8 may play an important role in arterial wall pathology.
_________________________
Lp(a) = lipoprotein(a)
HUVEC = human umbilical vein endothelial cells
Tomas Ganz, PhD, MD
Systemic infection or inflammation causes a decrease in intestinal iron absorption and impairs the release of recycled iron from macrophages. Decreased availability of iron may deny this essential element to invading pathogens and may inhibit their multiplication and other metabolic processes but also results in anemia of chronic disease. This article reviews recent discoveries that shed light on the regulation of iron metabolism during infection and iron overload, and point to the central role of a newly discovered peptide, hepcidin. Evidence to date indicates that hepcidin is a negative regulator of intestinal iron absorption, placental iron transport, and the release of iron from macrophages that recycle iron from senescent red cells. It may also be the central mediator of iron sequestration during infections and inflammatory states and the mediator of anemia of chronic disease. Rapid progress in this area is a good example of the beneficial effects of improvements in peptide analysis and chemistry, advances in genomics, and the increasing use of transgenic mice to determine the function of newly discovered genes and proteins.
Liat Nadav, MD, Benjamin Geiger, PhD and Ben-Zion Katz, PhD
Joseph D. Rosenblatt, MD, Seung-Uon Shin, PhD, Hovav Nechustan, MD, PhD, Kyung Hee Yi, BSc and Khaled Tolba, MD
Jacob Cohen, MSc, Lia Supino-Rosin, MSc, Eran Barzilay, BSc, Ronit Eisen-Lev, DMD, Moshe Mittelman, MD and Drorit Neumann, PhD
Yair Herishanu, MD and Shlomo Berliner, MD, PhD
Gabriel S. Breuer, MD, David Raveh, MD, Bernard Rudensky, PhD, Raina Rosenberg, MD, Rose Ruchlemer, MD and Jonathan Halevy, MD