The Bio-PIN: a concept to improve biobanking.

A new biobanking method is proposed, wherein samples and associated data would be deposited anonymously and labelled using a PIN code that is produced on the basis of personal biological characteristics, such as single nucleotide polymorphisms. The code would be the 'Bio-PIN' to uniquely distinguish the sample depositors, plus their samples and data. This method could help to diminish several long-discussed ethical, legal and societal problems in biobanking regarding privacy, informed consent, autonomy, data security and public trust.

Lifetime probabilities of hematopoietic stem cell transplantation in the U.S.

Healthcare policies regarding hematopoietic stem cell transplantation (HSCT) must address the need for the procedure as well as the availability of stem cell sources: bone marrow, peripheral blood, or umbilical cord blood (UCB). However, data with respect to the lifetime probability of undergoing HSCT are lacking. This study was undertaken to estimate the latter probability in the United States (U.S.), depending on age, sex, and race. We used data from the Center for International Blood and Marrow Transplant Research, the U.S. Surveillance, Epidemiology and End Results Program, and the U.S. Census Bureau and calculated probabilities as cumulative incidences. Several scenarios were considered: assuming current indications for autologous and allogeneic HSCT, assuming universal donor availability, and assuming broadening of HSCT use in hematologic malignancies. Incidences of diseases treated with HSCT and of HSCTs performed increase with age, rising strongly after age 40. Among individuals older than 40, incidences are higher for men than for women. The lifetime probabilities of undergoing HSCT range from 0.23% to 0.98% under the various scenarios. We conclude that, given current indications, the lifetime probability of undergoing autologous or allogeneic HSCT is much higher than previously reported by others and could rise even higher with increases in donor availability and HSCT applicability.

Antisense inhibition of hyaluronan synthase-2 in human articular chondrocytes inhibits proteoglycan retention and matrix assembly.

In order to define the role of cell-associated hyaluronan in cartilage matrix retention, human articular chondrocytes as well as cartilage slices were treated with phosphorothioate oligonucleotides comprised of sequence antisense to the mRNA of human HA synthase-2 (HAS-2). As a prerequisite for these studies, it was necessary to determine which HA synthase (HAS), of three separate human genes capable of synthesizing HA, designated HAS-1, HAS-2, or HAS-3, is primarily responsible for HA synthesis in human articular chondrocytes. The copy number of each HAS mRNA expressed in cultured human articular chondrocytes was determined using quantitative (competitive) reverse transcription-polymerase chain reaction (RT-PCR). Only HAS-2 and HAS-3 mRNA expression was detected. The level of HAS-2 mRNA expression was 40-fold higher than that of HAS-3. Cultures of human articular chondrocytes and cartilage tissue slices were then transfected with HAS-2-specific antisense oligonucleotides. This treatment resulted in time-dependent inhibition of HAS-2 mRNA expression, as measured by quantitative RT-PCR, and a significant loss of cell-associated HA staining. Sense and reverse HAS-2 oligonucleotides showed no effect. The consequences of reduced HA levels (due to HAS-2 antisense inhibition) were a decrease in the diameter of the cell-associated matrix and a decreased capacity to retain newly synthesized proteoglycan. These results suggest that HA synthesized by HAS-2 plays a crucial role in matrix assembly and retention by human articular chondrocytes.

Antisense inhibition of chondrocyte CD44 expression leading to cartilage chondrolysis.

OBJECTIVE: To better define critical functions of the hyaluronan receptor CD44 in cartilage. METHODS: Articular chondrocytes and cartilage tissue slices were treated with CD44 sequence-specific antisense phosphorothioate oligonucleotides. CD44 expression was probed by immunofluorescence microscopy, enzyme-linked immunosorbent assay, and Western blotting. RESULTS: Antisense oligonucleotides demonstrated a dose- and time-dependent inhibition of CD44 protein expression; negative controls showed no effect. Similar to osteoarthritic cartilage, antisense-treated cartilage slices displayed a near-total loss of stainable proteoglycan-rich matrix. CONCLUSION: CD44 expression is needed for maintenance of cartilage homeostasis.

Antisense oligonucleotides, a novel tool for the control of cytokine effects on human cartilage. Focus on interleukins 1 and 6 and proteoglycan synthesis.

OBJECTIVE: To prevent the negative effects of interleukin-1 (IL-1) and IL-1-induced IL-6 on cartilage proteoglycan (PG) synthesis, we used an antisense oligonucleotide (ASO) specific for IL-6 messenger RNA (mRNA) to inhibit IL-6 production. METHODS: Explants of human articular cartilage were cultured in the presence or absence of IL-6-ASO, IL-1, and exogenous IL-6. As metabolic parameters, cartilage production of IL-6 was determined in the B9 bioassay and PG as incorporation of 35SO4. RESULTS: The IL-6-ASO prevented IL-1-induced production of IL-6 in the cartilage explants, as well as IL-1-induced inhibition of PG synthesis. This inhibition was restored, despite the presence of IL-6-ASO, when exogenous IL-6 was added. A control ASO (not specific for IL-6 mRNA) was not effective. CONCLUSION: The IL-6-ASO used can penetrate the extracellular matrix of articular cartilage, enter the chondrocytes, and inhibit the IL-1-induced production of IL-6. Furthermore, IL-6-ASO can prevent the IL-1-induced inhibition of cartilage PG synthesis. The effect of exogenous IL-6 shows that IL-1 requires IL-6 for inhibition of PG synthesis.

Cytokines and proteoglycans.

Cytokines play an important regulatory role in the metabolism of proteoglycans. Proteoglycans are found in plasma membranes, but predominantly in the extra-cellular matrix. In the latter they are quantitatively and qualitatively essential components. Especially in a tissue like cartilage without any blood vessels, the cells are dependent on cytokines for the communication among themselves in the extra-cellular matrix and also for communication with the 'outside world'. Various cytokines have been found to be able to penetrate the extra-cellular matrix and inhibit, respectively stimulate the proteoglycan synthesis. Also, the degradation of proteoglycans can be stimulated, respectively inhibited by several cytokines. In addition, some cytokines have been found which regulate the effects of the other cytokines. With respect to proteoglycan metabolism a complex cytokine network is emerging. Furthermore it is becoming increasingly clear that proteoglycans are connected to the cytokine network by their own bioactive functions. First, they possibly possess cytokine activities themselves. Second, they can function as receptors, protectors, inactivators and storage ligands for cytokines. So the proteoglycans are clearly involved in the feedback signalling from the extra-cellular matrix to the cells that are synthesizing this extra-cellular matrix. Together with agonistic or antagonistic cytokines they are involved in the regulation of proteoglycan turnover during balanced or unbalanced metabolism in normal, respectively pathological situations.

Elevated synovial fluid levels of interleukin-6 and tumor necrosis factor associated with early experimental canine osteoarthritis.

OBJECTIVE: To measure levels of cytokines, proteases, and glycosaminoglycans (GAG) in synovial fluid (SF) from the knees of animals with experimental osteoarthritis (OA) and from their contralateral (control) knees, and to compare and correlate these values with each other as well as with measures of proteoglycan synthesis in the corresponding articular cartilage. This study will help to identify cytokines of potential importance in the early stages of the development of OA. METHODS: OA was induced in 12 mature animals by sectioning the anterior cruciate ligament. After 3 months, SF from the operated and contralateral (control) knee joints was assayed for interleukin-6 (IL-6), tumor necrosis factor (TNF), IL-1, latent metalloproteinase, and sulfated GAG. Proteoglycan synthesis in the corresponding articular cartilage was also measured. RESULTS: IL-6 levels in SF from the operated joint compared with the control joint were significantly elevated in 11 of 12 animals. TNF levels were also elevated in 10 of 11 SF samples from operated joints, but to a lesser extent than those of IL-6. IL-1 and IL-1 inhibitors were undetectable in either the operated or control joint SF. The GAG concentration was elevated in SF from experimental OA joints. This elevation correlated with that of TNF, but not IL-6. There was no significant difference in the concentration of APMA-activatable metalloproteinase. The rate of proteoglycan synthesis was higher in the cartilage from the operated joint in 8 of 12 animals, and the mean rate of synthesis was significantly higher than in the control joint. There was a positive correlation between this increase in cartilage proteoglycan synthesis (operated versus control) and the increase in SF IL-6, but there was no correlation with the levels of TNF or GAG. CONCLUSION: This is the first study of SF levels of cytokines in early experimental OA. Our results show surprisingly high levels of IL-6 in operated joints, where the cytokine could act directly on the chondrocytes, and thus play a role in mediating their responses to cartilage injury.

Cytokines and proteoglycans.

Cytokines play an important regulatory role in the metabolism of proteoglycans. Proteoglycans are found in plasma membranes, but predominantly in the extra-cellular matrix. In the latter they are quantitatively and qualitatively essential components. Especially in a tissue like cartilage without any blood vessels, the cells are dependent on cytokines for the communication among themselves in the extra-cellular matrix and also for communication with the 'outside world'. Various cytokines have been found to be able to penetrate the extra-cellular matrix and inhibit, respectively stimulate the proteoglycan synthesis. Also, the degradation of proteoglycans can be stimulated, respectively inhibited by several cytokines. In addition, some cytokines have been found which regulate the effects of the other cytokines. With respect to proteoglycan metabolism a complex cytokine network is emerging. Furthermore it is becoming increasingly clear that proteoglycans are connected to the cytokine network by their own bioactive functions. First, they possibly possess cytokine activities themselves. Second, they can function as receptors, protectors, inactivators and storage ligands for cytokines. So the proteoglycans are clearly involved in the feedback signalling from the extra-cellular matrix to the cells that are synthesizing this extra-cellular matrix. Together with agonistic or antagonistic cytokines they are involved in the regulation of proteoglycan turnover during balanced or unbalanced metabolism in normal, respectively pathological situations.

Role of TNF alpha, in relation to IL-1 and IL-6 in the proteoglycan turnover of human articular cartilage.

In both young and old human articular cartilage explants, TNF alpha induced a concentration-dependent, reversible suppression of the proteoglycan (PG) synthesis. Young cartilage was more sensitive to TNF alpha than old cartilage: 50% suppression of PG synthesis was reached at a TNF alpha concentration of 5 U/ml for young and 30 U/ml for old cartilage, whereas at 10(3) U/ml the PG synthesis of young cartilage was blocked and that of old cartilage suppressed by 80%. These inhibition levels of PG synthesis resulted in 25% PG depletion of the explants after 8 days of culture. The release of cartilage PG was not enhanced. TNF alpha induced no detectable amounts of IL-1 (less than 0.01 U) in young or old cartilage but did induce IL-6 production. The induced amounts of IL-6 were higher in young than in old cartilage but no dose-dependency was evident. Antibodies to neither IL-1 nor IL-6 had any influence on the TNF alpha-induced suppression of PG synthesis. The combination of TNF alpha and IL-1 led to an additive inhibition of PG synthesis which had no relationship to induced IL-6. TNF alpha was about 100-fold less active than IL-1.

Interleukin-1-induced interleukin-6 is required for the inhibition of proteoglycan synthesis by interleukin-1 in human articular cartilage.

Cartilage from normal controls, patients with osteoarthritis, and patients with rheumatoid arthritis produced no interleukin-6 (IL-6) in culture. However, IL-1 induced massive production of IL-6 (up to 135 ng/ml) in cartilage from all 3 sources, in a dose-dependent manner (in some cases, a peak value was reached). The levels of induced IL-6 were similar to those found in rheumatoid arthritis synovial fluid. At IL-1 concentrations that induced almost complete inhibition of proteoglycan (PG) synthesis, IL-6 production could still be increased considerably. Exogenous IL-6 inhibited PG synthesis by up to 25%. IL-1-induced inhibition of PG synthesis was reversed by antibodies against recombinant human IL-6. These results suggest that IL-6 is required for the IL-1-induced inhibition of PG synthesis.

The effect of human interleukin 1 on proteoglycan metabolism in human and porcine cartilage explants.

Human interleukin 1 (IL-1), up to 100 pg/ml, causes a decrease of the proteoglycan content of human (old and young) as well as porcine cartilage explants, without stimulating the proteoglycan release from the cartilage. The proteoglycan depletion is stronger in young than in old human cartilage and stronger in human than in porcine cartilage. The proteoglycan synthesis is considerably more inhibited by IL-1 in young than in old human cartilage. Our data suggest that an IL-1 induced inhibition of the proteoglycan synthesis, rather than a stimulation of proteoglycan breakdown causes the proteoglycan depletion of the cartilage. The data furthermore suggest a clear difference between young and old human cartilage, with respect to their sensitivity for IL-1. IL-1 in a concentration of 500 pg/ml causes in all 3 kinds of cartilage explants chondrocyte damage that might be relevant in the cartilage destruction during rheumatoid arthritis.

The influence of 2-oxoglutarate on the activity of prolyl 4-hydroxylase.

1. Treatment of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with 2-oxoglutarate in the absence of added Fe2+ for 10 s causes partial inactivation of the enzyme which is not reversed by subsequent addition of Fe2+. It appears that 2-oxoglutarate prevents loss of enzyme-bound iron and prevents access of added iron to its binding site. 2. For optimal enzyme activity the enzyme should be preincubated for 15 s with Fe2+ (5 microM). 3. Under turnover conditions prolyl 4-hydroxylase does not release iron. 4. The inactivation brought about by pre-incubation with 2-oxoglutarate and O2 in the absence of ascorbate is partly reversed by removal of 2-oxoglutarate. 5. It is proposed that dead-end complex formation with 2-oxoglutarate is responsible for the inactivation of the enzyme by 2-oxoglutarate in the absence of either ascorbate or Fe2+. 6. Optimal enzyme activity is obtained if the reactants are added to the reaction medium in the following order: enzyme, iron and ascorbate in any order, then after at least 15 s 2-oxoglutarate and finally (Pro-Pro-Gly)5 to start the reaction.

Dissociation and reassociation of prolyl 4-hydroxylase subunits after cross-linking of monomers.

1. Incubation of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with H2O2 leads to a decrease of 50% in the specific activity of enzyme tetramers, followed by dissociation into inactive dimers in which the monomers are covalently cross-linked by S-S bridge formation. 2. Incubation of the enzyme with K3Fe(CN)6 leads to a comparable decrease in activity of enzyme tetramers. Addition of urea leads to dissociation into inactive dimers with similarly cross-linked monomers. 3. Removal of the dissociating agent leads to reassociation of cross-linked dimers to tetramers and to about 50% reactivation. The enzyme is further reactivated by preincubation with dithiothreitol. 4. Dissociation of the enzyme with dithiothreitol, urea or LiCl, or at low pH (4.15) produces inactive monomers, which could not be reassociated.