Manufacturing Mesenchymal Stromal Cells for the Treatment of Graft-versus-Host Disease: A Survey among Centers Affiliated with the European Society for Blood and Marrow Transplantation.

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results.

Osteocalcin positive mononuclear cells are associated with the severity of aortic calcification.

OBJECTIVE: To assess the association of circulating bone marrow-derived osteo-progenitors with vascular calcification in mouse models and patients with peripheral artery disease. METHODS: We estimated the percentage of circulating mononuclear cells expressing osteocalcin in 2 mouse models of aortic calcification developed in osteoprotegerin-deficient mice (OPG(-/-)) using flow cytometry. Aortic calcification was assessed in mice principally by a bioassay of harvested aortas. In patients with peripheral artery disease osteocalcin-positive cells (estimated by flow cytometry) were related to aortic calcification volume assessed from computed tomography. RESULTS: The amount of extractable aortic calcium was increased in both mouse models used in comparison to controls. The percentage of circulating mononuclear cells expressing osteocalcin was correlated to the amount of extractable aortic calcium in male (r=0.525, p=0.02) and female OPG(-/-) (r=0.564, p=0.02) mice and also in animals in which calcification was accelerated using calcitriol (r=0.64, p=0.01). Patients with more severe aortic calcification had a greater percentage of circulating OCN(+) MNCs (median 4.07%, IQR 3.76-4.39, n=12) than those with less severe aortic calcification (median 3.10%, IQR 2.32-3.60, n=11, p=0.05). CONCLUSIONS: This study demonstrates that aortic calcification can be robustly quantified in 2 mouse models. In these models and patients with peripheral artery disease circulating osteocalcin positive mononuclear cells are associated with the severity of aortic calcification.

Osteoprotegerin, vascular calcification and atherosclerosis.

The association of bone pathologies with atherosclerosis has stimulated the search for common mediators linking the skeletal and the vascular system. Since its initial discovery as a key regulator in bone metabolism, osteoprotegerin (OPG) has become the subject of intense interest for its role in vascular disease and calcification. Studies in vitro and in animal models suggest that OPG inhibits vascular calcification. Paradoxically however, clinical studies suggest that serum OPG levels increase in association with vascular calcification, coronary artery disease, stroke and future cardiovascular events. This has led to an extensive debate on the potential of OPG as a biomarker of vascular disease. However the exact significance and mechanisms by which this bone-regulatory protein influences cardiovascular pathophysiology is still unclear. The need for a more complete picture is being addressed in increasing valuable research indicating OPG as not only a marker but also a mediator of vascular pathology modulating osteogenic, inflammatory and apoptotic responses. By integrating the results of recent experimental research, animal models and clinical studies, this review summarises the present understanding of the role of OPG in vascular disease and calcification.

Role of homocysteine in aortic calcification and osteogenic cell differentiation.

BACKGROUND: The role of homocysteine in atherosclerosis is unclear. We examined the relationship between plasma homocysteine and infrarenal aortic calcification, the presence of homocysteine in human atheroma and the influence of homocysteine on osteogenic differentiation in vitro. METHODS AND RESULTS: In 194 patients with symptomatic peripheral artery disease or abdominal aortic aneurysm, fasting plasma total homocysteine was independently associated with the severity of infrarenal aortic calcification measured by Computer Tomography Angiography (odds ratio 1.91, 95% confidence interval 1.17-3.21 for calcification >or=median). Homocysteine was identified in all 60 atheroma biopsies from 16 patients undergoing endarterectomy, and concentrations were significantly greater in the calcified biopsies (p=0.003). In vitro studies demonstrated that 100 micromol/L homocysteine doubled the calcium deposition by mesenchymal stem cells during 16 days incubation in osteogenic medium (74+/-4 compared to 42+/-5 microg calcium/well without homocysteine, p<0.001). Homocysteine also stimulated monocytic THP1 cells to promote aortic smooth muscle cell calcification as evidenced by significant higher calcium deposition and alkaline phosphatase activity compared to incubation without homocysteine (p

Iron-induced oxidative stress in haemodialysis patients: a pilot study on the impact of diabetes.

BACKGROUND: Administration of intravenous iron preparations in haemodialysis patients may lead to the appearance of non-transferrin bound iron which can catalyse oxidative damage. We investigated this hypothesis by monitoring the oxidative stress of haemodialysis patients and the impact of iron and diabetes mellitus herein. MATERIALS AND METHODS: Baseline values of serum iron and related proteins, transferrin glycation, non-transferrin bound iron, antioxidant capacity and lipid peroxidation (malondialdehyde) of 11 haemodialysis patients (six non-diabetic and five type 2 diabetes) were compared to those of non-haemodialysis control subjects (non-diabetic and type 2 diabetes). Changes in these parameters were monitored during haemodialysis before and after iron administration. RESULTS: Baseline values of malondialdehyde correlated with ferritin concentration (r = 0.664, P = 0.036) and were elevated to the same extent in non-diabetic and diabetic haemodialysis patients (median of 1.09 compared to 0.60 mumol/l in control persons, P < 0.02). After iron infusion, transferrin saturation increased more markedly in non-diabetic subjects from 28% to 185% vs. from 33% to 101% in diabetic patients (P = 0.008). This increase was accompanied by the appearance of non-transferrin bound iron (5.91 +/- 1.33 micromol/l), a loss in plasma iron-binding antioxidant capacity and a further increase in malondialdehyde which was more pronounced in diabetic patients (from 0.93 +/- 0.30 micromol/l to 2.21 +/- 0.69 micromol/l vs. from 1.21 +/- 0.42 micromol/l to 1.86 +/- 0.56 micromol/l in the non-diabetic subjects, P = 0.046). CONCLUSIONS: In haemodialysis patients, higher lipid peroxidation is determined by higher body iron stores. The increase induced by iron infusion is accompanied by a loss in iron-binding antioxidant capacity and is more pronounced in diabetes mellitus.

Bone marrow-derived cells and arterial disease.

This article reviews the association between bone and artery disease, with particular relevance to progenitor cells. The review was based on insight gained by analysis of previous publications and on-going work by the authors. A large number of studies have demonstrated a correlation between bone pathology, particularly osteoporosis, and atherosclerosis. In this review we highlight the particular aspect of bone marrow progenitor cells in the bone-artery link. Progenitor cells, primarily those believed to give rise to endothelial cells, have been inversely correlated with atherosclerosis severity and risk factors. Therapeutic approaches aimed at manipulating progenitor cells in revascularization and vascular repair have demonstrated some promising results. Subtypes of progenitor cells have also been linked with vascular pathology, however, and further studies are required to assess relative beneficial and pathologic effects of bone marrow-derived progenitors. Further understanding of the link between bone and artery pathophysiology is likely to be of significant value in developing new therapies for vascular disease.

Osteoprotegerin upregulates endothelial cell adhesion molecule response to tumor necrosis factor-alpha associated with induction of angiopoietin-2.

OBJECTIVE: Osteoprotegerin (OPG) and osteopontin (OPN) have been identified within unstable atherosclerosis and circulating concentrates have been linked to cardiovascular events. We studied the influence of OPG and OPN on endothelial adhesion molecule expression and monocyte binding. METHODS: Resting or tumor necrosis factor (TNF-alpha) activated human endothelial cells were incubated with OPG (0, 0.5, 5, and 10 ng/mL) or OPN (0, 2.5, 10 and 50 nmol/L). The expression of endothelial genes and proteins was investigated with the Oligo GEArray microarray series, multiplexed gene expression analysis, flow cytometry, ELISA and immunohistochemistry. Monocyte-binding studies were carried out using fluorescently labeled THP-1 cells and analysed by flow cytometry. RESULTS: OPG but not OPN stimulated a dose-dependent increase in the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin by endothelial cells in the presence of TNF-alpha (p

Iron-binding antioxidant capacity is impaired in diabetes mellitus.

Increased lipid peroxidation contributes to diabetic complications and redox-active iron is known to play an important role in catalyzing peroxidation reactions. We aimed to investigate if diabetes affects the capacity of plasma to protect against iron-driven lipid peroxidation and to identify underlying factors. Glycemic control, serum iron, proteins involved in iron homeostasis, plasma iron-binding antioxidant capacity in a liposomal model, and non-transferrin-bound iron were measured in 40 type 1 and 67 type 2 diabetic patients compared to 100 nondiabetic healthy control subjects. Iron-binding antioxidant capacity was significantly lower in the plasma of diabetic subjects (83 +/- 6 and 84 +/- 5% in type 1 and type 2 diabetes versus 88 +/- 6% in control subjects, p < 0.0005). The contribution of transferrin, ceruloplasmin, and albumin concentrations to the iron-binding antioxidant capacity was lost in diabetes (explaining only 4.2 and 6.3% of the variance in type 1 and type 2 diabetes versus 13.9% in control subjects). This observation could not be explained by differences in Tf glycation, lipid, or inflammatory status and was not associated with higher non-transferrin-bound iron levels. Iron-binding antioxidant capacity is decreased in diabetes mellitus.

A novel method to quantify in vivo transferrin glycation: applications in diabetes mellitus.

BACKGROUND: In vitro glycation of transferrin leads to increased oxidative stress by impairing iron-binding antioxidant capacity. The aim of this study is to develop a method to evaluate in vivo transferrin glycation in diabetes. METHODS: We adapted the nitroblue tetrazolium assay to measure in micro-well plates the fructosamine content of transferrin isolated from serum by immunocomplexation. RESULTS: Introduction of the immunocomplexation step did not affect the analytical performance of the fructosamine measurement and analytical variability was lower than 7%. The diabetic group (n=107) had significantly higher transferrin glycation (1.39+/-1.12 versus 0.79+/-1.09 micromol fructosamine/g transferrin in the non-diabetic group, n=91, p<0.0005) and this was most pronounced in type 1 diabetes (1.95+/-1.02 versus 1.06+/-1.04 micromol fructosamine/g transferrin in type 2, p<0.0005). Transferrin glycation was associated with parameters of glycaemic control but did not correlate with serum iron or total iron-binding capacity. Total iron-binding capacity was lower in type 1 diabetes (63+/-9 versus 69+/-12 micromol/l in type 2, p<0.05) and was mainly determined by transferrin concentration. CONCLUSIONS: These results indicate that the adapted nitroblue tetrazolium assay combined with immunocomplexation of serum transferrin is suitable to detect differences in in vivo transferrin glycation between non-diabetic, type 1 and type 2 diabetic subjects.

Impact of diabetes mellitus on the relationships between iron-, inflammatory- and oxidative stress status.

BACKGROUND: Diabetes is an inflammatory condition associated with iron abnormalities and increased oxidative damage. We aimed to investigate how diabetes affects the interrelationships between these pathogenic mechanisms. METHODS: Glycaemic control, serum iron, proteins involved in iron homeostasis, global antioxidant capacity and levels of antioxidants and peroxidation products were measured in 39 type 1 and 67 type 2 diabetic patients and 100 control subjects. RESULTS: Although serum iron was lower in diabetes, serum ferritin was elevated in type 2 diabetes (p = 0.02). This increase was not related to inflammation (C-reactive protein) but inversely correlated with soluble transferrin receptors (r = - 0.38, p = 0.002). Haptoglobin was higher in both type 1 and type 2 diabetes (p < 0.001) and haemopexin was higher in type 2 diabetes (p < 0.001). The relation between C-reactive protein and haemopexin was lost in type 2 diabetes (r = 0.15, p = 0.27 vs r = 0.63, p < 0.001 in type 1 diabetes and r = 0.36, p = 0.001 in controls). Haemopexin levels were independently determined by triacylglycerol (R(2) = 0.43) and the diabetic state (R(2) = 0.13). Regarding oxidative stress status, lower antioxidant concentrations were found for retinol and uric acid in type 1 diabetes, alpha-tocopherol and ascorbate in type 2 diabetes and protein thiols in both types. These decreases were partially explained by metabolic-, inflammatory- and iron alterations. An additional independent effect of the diabetic state on the oxidative stress status could be identified (R(2) = 0.5-0.14). CONCLUSIONS: Circulating proteins, body iron stores, inflammation, oxidative stress and their interrelationships are abnormal in patients with diabetes and differ between type 1 and type 2 diabetes.

Time course of oxidative stress status in the postprandial and postabsorptive states in type 1 diabetes mellitus: relationship to glucose and lipid changes.

OBJECTIVE: The aim of this study was to compare oxidative stress status (OSS) with blood glucose and lipid changes during the fasting, postprandial and postabsorptive phases in type 1 diabetes mellitus. METHODS: Twenty-three patients on intensive insulin treatment received a standard fat-rich breakfast and lunch. OSS was monitored at fasting (F), just after the post-breakfast glycemia peak (BP) (identified by continuous subcutaneous glucose monitoring), 3.5-h post-breakfast (B3.5), just after the post-lunch peak (LP), just after the post-lunch dale (LD) and 5 hours after lunch (L5). RESULTS: Whereas whole blood glutathione and plasma protein thiols increased in the postprandial period (from 6.52 +/- 1.20 (F) to 7.08 +/- 1.45 micromol/g Hb (BP), p = 0.005), ascorbate decreased gradually from 44 +/- 17 (F) to 39 +/- 19 micromol/L (LD), p = 0.015. Retinol and alpha-tocopherol also decreased from 27.1 +/- 7.0 (F) to 25.3 +/- 5.2 micromol/L (BP), p = 0.005. Uric acid decreased later, from 213 +/- 77 (BP) to 204 +/- 68 micromol/L (B3.5), p = 0.01, but then increased in LP (231 +/- 70 micromol/L) and LD to values higher than F (215 +/- 64, micromol/L, p = 0.01). Malondialdehyde increased gradually from 1.02 +/- 0.36 (F) to a maximum of 1.14 +/- 0.40 micromol/L (LP). In the postabsorptive phase (L5) all parameters except for thiols reverted to fasting concentrations. CONCLUSIONS: In type 1 diabetes lipid peroxidation increases during the postprandial phase in parallel to glucose and triglyceride changes. Blood antioxidants, however, followed diverse patterns of change.

Cell-mediated LDL oxidation: the impact of transition metals and transferrin.

Activated monocytes release oxygen radicals by respiratory burst and oxidative damage can be accelerated by transition metals. We investigated the cell-mediated and metal-catalysed in vitro oxidation of low-density lipoproteins (LDL), as well as the impact of the metal-binding protein transferrin (Tf). LDL oxidation was measured by monitoring the increase in fluorescence (350/440 nm excitation/emission). Maximal respiratory burst by U937 cells was achieved after 96 h differentiation with retinoic acid and dihydroxyvitamin D3 followed by stimulation with opsonised zymosan. Addition of activated cells resulted in the LDL oxidation, even in the absence of transition metals. Moreover, activated cells greatly enhanced metal-catalysed oxidative modifications, especially in the presence of copper. By binding metals, Tf was able to strongly impair this process. In conclusion, by generating oxygen radicals, activated U937 cells were able to oxidise LDL. The oxidising process was most pronounced in the presence of copper and could be blocked by Tf.

Effects of in vitro glycation on Fe3+ binding and Fe3+ isoforms of transferrin.

BACKGROUND: In diabetes, protein function is altered by glycation, but the impact on the Fe3+ binding and antioxidant functions of transferrin (Tf) is largely unknown. The aim of the present study was to investigate the effects of glycation on the distribution of Fe3+ on the two Fe3+ -binding sites of Tf. METHODS: In vitro glycation of Tf was accomplished by preincubation with glucose for 14 days. Tf was loaded with Fe3+ compounds to achieve theoretical Tf Fe3+ saturations of 32%, 64%, and 96% (monitored by spectrophotometry). Fe3+ -Tf isoforms were separated by isoelectric focusing. RESULTS: Fe3+ binding was highest when Tf was incubated with Fe:nitrilotriacetic acid and reached a steady state overnight. Increasing the Fe3+ load led to a shift of isoform profile toward the diferric form (Fe2-Tf): in freshly prepared Tf, Fe2-Tf represented 6%, 30%, and 66% of all isoforms at 32%, 64%, and 96% theoretical Fe3+ saturation, respectively. Fe3+ was equally distributed to the monoferric Tf forms with Fe3+ bound to the amino (Fe1N-Tf) and carboxy termini (Fe1C-Tf). Glycation decreased binding of Fe3+ to Tf (monitored at 450 nm). At low theoretical Fe3+ saturation (32%), glycation increased the mean (SD) proportion of Fe2-Tf: 18 (3)% in the presence of 33.3 mmol/L glucose vs 12 (4)% with 0 mmol/L glucose (P = 0.01). In contrast, at 96% theoretical Fe3+ saturation, Fe2-Tf decreased linearly with increasing glycation (r = 0.97; P = 0.008). Preincubation, independent of glycation, favored the Fe1N-Tf isoform at 64% theoretical Fe3+ saturation [27 (0.7)% vs 23 (1.1)% of the Fe1C-Tf isoform; P = 0.009]. CONCLUSIONS: Glycation impairs Fe3+ binding and affects Fe3+ -Tf isoform distribution depending on concentration. The diagnostic implications of these results need further elucidation in clinical studies.

Evolution of serum alpha-tocopherol in the postprandial and postabsorptive phases in type 1 diabetes mellitus.

Blood glucose, lipids, alpha-tocopherol, and malondialdehyde were monitored in type 1 diabetes mellitus patients for 8 hours after a standard fat-rich breakfast and lunch. Although glucose and triglycerides increased, alpha-tocopherol decreased and malondialdehyde increased in the postprandial phase. In the postabsorptive phase values returned to fasting levels. These results point to the possible relevance of postprandial alpha-tocopherol depletion and lipid peroxidation to an increased cardiovascular risk in these patients.

Transferrin modifications and lipid peroxidation: implications in diabetes mellitus.

Free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation. One of the most important mechanisms of antioxidant defense is thus the sequestration of iron in a redox-inactive form by transferrin. In diabetes mellitus, increased oxidative stress and lipid peroxidation contribute to chronic complications but it is not known if this is related to abnormalities in transferrin function. In this study we investigated the role of transferrin concentration and glycation. The antioxidant capacity of apotransferrin to inhibit lipid peroxidation by iron-binding decreased in a concentration-dependent manner from 89% at > or = 2 mg/ml to 42% at 0.5 mg/ml. Pre-incubation of apotransferrin with glucose for 14 days resulted in a concentration-dependent increase of glycation: 1, 5 and 13 micromol fructosamine/g transferrin at 0, 5.6 and 33.3 mmol/l glucose respectively, p < 0.001. This was accompanied by a decrease in the iron-binding antioxidant capacity of apotransferrin. In contrast, transferrin glycation by up to 33.3 mmol/l glucose did not affect chemiluminescence-quenching antioxidant capacity, which is iron-independent. Colorimetric evaluation of total iron binding capacity in the presence of an excess of iron (iron/transferrin molar ratio = 2.4) also decreased from 0.726 to 0.696 and 0.585mg/g transferrin after 0, 5.6 and 33.3 mmol/l glucose, respectively, p < 0.01. In conclusion, these results suggest that lower transferrin concentration and its glycation can, by enhancing the pro-oxidant effects of iron, contribute to the increased lipid peroxidation observed in diabetes.