Matthias Theobald

Molecular design of the C alpha beta interface favors specific pairing of introduced TCR alpha beta in human T cells

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRα- and TCRβ-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRα- and TCRβ-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Cα and Cβ domains and are related to each other by both a complementary steric interaction analogous to a “knob-into-hole” configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged “hole-into-knob” configuration. We show that this inversion in the CαCβ interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

The combination of the biomarkers urinary C‐terminal telopeptide of type II collagen, serum cartilage oligomeric matrix protein, and serum chondroitin sulfate 846 reflects cartilage damage in hemophilic arthropathy

OBJECTIVEnHemophilic arthropathy, with characteristics of inflammatory (rheumatoid arthritis) and degenerative (osteoarthritis) joint damage, occurs at an early age, is associated with minor comorbidity, and is restricted to 3 pairs of large joints. The aim of this study was to determine whether commonly used serum and/or urinary biomarkers of cartilage and bone turnover for which assay kits are commercially available are associated with the severity of joint damage in patients with various degrees of hemophilic arthropathy and, thus, whether this disease could be useful in the identification and evaluation of such biomarkers.nnnMETHODSnBlood and urine samples were collected from 36 patients with various degrees of hemophilic arthropathy. Commercially available assays for the most frequently investigated serum and urine biomarkers were performed: urinary C-terminal telopeptide of type I collagen (CTX-I), urinary CTX-II, serum CTX-I, serum CTX-II, serum cartilage oligomeric matrix protein (COMP), serum cartilage cleavage products C1,2C and C2C, and serum chondroitin sulfate 846 (CS-846). Radiographs of the ankles, knees, and elbows in all patients were evaluated for the degree of joint damage according to the Pettersson score, which is based on cartilage and periarticular bone changes and is specific for hemophilic arthropathy.nnnRESULTSnUrinary CTX-II, serum C1,2C, and serum CS-846 levels correlated with the overall Pettersson score and with the joint space narrowing component. Regression analysis showed that combined indexes of different markers increased the degree of correlation for the combination of urinary CTX-II, serum COMP, and serum CS-846. Bone-specific markers (urinary/serum CTX-I and serum C1,2C) did not correlate with specific bone-related items of the Pettersson score (osteoporosis and erosions).nnnCONCLUSIONnThese results support the idea that a combination of biomarkers relates significantly better to the severity of joint damage than do individual biomarkers. The combination of urinary CTX-II, serum COMP, and serum CS-846 correlated best with the degree of arthropathy. Because of its specific characteristics and restricted involvement, hemophilic arthropathy may prove useful in the screening of newly developed biomarkers of joint damage.

Very rapid clearance after a joint bleed in the canine knee cannot prevent adverse effects on cartilage and synovial tissue

OBJECTIVEnJoint bleeding leads to joint destruction. In vitro exposure of human and canine cartilage to blood results in long-lasting severe adverse changes in cartilage. An in vivo joint haemorrhage in the canine knee joint demonstrates similar adverse effects although significantly less outspoken. As a possible explanation for this discrepancy, we studied the clearance rate of blood from the canine knee joints.nnnMETHODSnBlood was injected into the knee joint of Beagle dogs either 48 h, 24h or 15 min before termination. The amount of red blood cells (RBC) and white blood cells (WBCs) present in the joint cavity was determined. Chondrocyte activity and cartilage matrix integrity as well as cartilage destructive activity of synovial tissue were determined biochemically. Additionally, synovial tissue was analyzed by use of histochemistry.nnnRESULTSnThe amount of blood was decreased to <5% within 48 h. Within this time period the cartilage was negatively affected and the synovial tissue showed cartilage destructive activity. Evaluation of the synovial tissue 15 min post-injection revealed countless numbers of intact RBC that were almost completely disappeared after 48 h without significant recruitment of macrophages.nnnCONCLUSIONSnBlood is cleared very rapidly from the canine knee joint, but already has adverse effects on both cartilage and synovial tissue within that short time span. This rapid clearance can play a role in the discrepancy between long-term in vitro and in vivo effects of blood-induced joint damage since more than 10% v/v blood present for at least 48 h is needed to induce long-term adverse effects in vitro.

EGF and HGF levels are increased during active HBV infection and enhance survival signaling through extracellular matrix interactions in primary human hepatocytes

The hepatitis B virus (HBV) is a major causative agent of chronic liver disease and subsequent liver cirrhosis worldwide. The reduced sensitivity of virus‐infected liver cells to apoptosis may play a role in the failure to remove virus‐infected cells and eventually promote viral chronicity. The purpose of our study was to investigate whether survival factors induced during compensatory liver regeneration may protect hepatocytes against apoptosis. We evaluated the serum levels of hepatocyte growth factor (HGF) and epidermal growth factor (EGF) in HBV‐infected patients and found significant increases in HGF and EGF in patients with active virus infection. In primary human hepatocytes we show that HGF and EGF have a protective effect against CD95‐mediated apoptosis and cytotoxic T‐cell killing. Simultaneous treatment with both regeneration factors enhanced the cytoprotective effect. The PI 3‐K/Akt kinase inhibitor, wortmannin, and the STAT3 pathway inhibitor, Tyrphostin AG490, both effectively attenuated the cytoprotective effect of HGF and EGF. Furthermore, we show an EGF/HGF‐dependent upregulation of β1‐integrin chains, increased adhesion to extracellular matrix and an increase in focal adhesions, suggesting outside‐in signaling from the extracellular matrix as an additional cytoprotective mechanism. Our study demonstrates that HGF and EGF can interfere with CD95‐mediated apoptosis and the action of cytotoxic T‐cells through multiple mechanisms in human hepatocytes. Together our results argue that a survival mileau generated by activation of liver regeneration factors may be a risk factor for establishing viral persistence.

The non-ionic surfactant poloxamer P188 does not prevent blood-induced cartilage damage

Ample evidence is gained over the past years, by both human in vitro and animal in vivo studies, that blood has a direct harmful effect on cartilage [1]. Although the observed effects after a first (or limited number of) joint bleed(s) will not be directly visible in clinical practice, they clearly will compromise the joint, limiting the regenerative capacity needed in case of additional stresses on the cartilage, such as loading or additional haemorrhages. The most comprehensible example of bloodinduced joint damage is seen in haemophilia. The repeated joint bleeds lead to joint damage within several years. This haemophilic arthropathy (HA) owing to recurrent haemarthroses is the main cause of morbidity in patients suffering from haemophilia. In addition, even despite the significant improvement of treatment with clotting factors, joint bleeds still occur. A recent study by Manco-Johnson in the New England Journal of Medicine clearly demonstrated this. As the editorial on this study stated [2], it will be impossible, owing to high costs, to treat all patients with haemophilia in such a way (complete clotting factor substitution) that joint bleeding will be banned completely. Therefore, a search of treatment modalities to prevent haemarthropathy is warranted. However, thus far there is no treatment at all for blood-induced joint damage. There is even hardly any research on possible treatment modalities. This might be because of the significant delay between the actual bleeding and the clinical outcome, the relative low incidence of severe haemophilia, and the relatively recent knowledge gained on the mechanisms of blood-induced joint damage [1]. As is demonstrated by several human in vitro studies, exposure of cartilage to blood results in adverse changes in chondrocyte activity, fully independent of synovial tissue and inflammatory mediators. Hydroxyl radicals are formed when hydrogen peroxide production by chondrocytes is increased upon stimulation by pro-inflammatory cytokines such as interleukin (IL)-1, originating from activated blood monocytes/macrophages as present in the blood within the joint after a haemarthros. Hydrogen peroxide reacts with haemoglobin-derived iron from damaged and phagocytosed red blood cells in the vicinity of chondrocytes, leading to the formation of radicals that induce cell death, as was shown among others by Transferase Biotin-dUTP Nick End Labeling (TUNEL) staining [3]. Inhibitors of radical formation and apoptosis were able to inhibit the harmful effects of blood on cartilage partially. Chondrocyte death leads to significant long-lasting disturbance of matrix turnover, because chondrocytes maintain cartilage matrix integrity on their own. Moreover, because of chondrocyte death, cartilage will be impaired in its regenerative capacity needed to recover from stresses from biomechanical and biochemical origin. This is because chondrocytes in adult cartilage hardly proliferate; the chondrocytes present have to do the job for a lifetime. For this reason, chondrocyte death has to be prevented as good as possible. In this respect, recent studies on the potential chondro-protective capacity of poloxamer 188 (P188 or pluronic) drew the attention. P188 is a poloxamer surfactant belonging to the class of water-soluable multi-block copolymers that have important surface-active properties. P188 is a tri-block copolymer having an average molecular weight of 8400 Da. It consists of two hydrophilic sites with a hydrophobic site in-between. This Correspondence: Nathalie W.D. Jansen, MSc, Rheumatology & Clin. Immunology, University Medical Center Utrecht, Room F.02.127, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Tel.: 31.88.7557569; fax: 31.30.2523741; e-mail: n.w.d.jansen@umcutrecht.nl

197 INTERLEUKIN-10 PROTECTS AGAINST BLOOD-INDUCED JOINT DAMAGE

Purpose: Joint bleeds occur frequently in patients suffering from haemophilia or can occur as a consequence of joint trauma or major joint surgery. Even a limited number of bleeds has been demonstrated to lead to severe joint damage in time. Small amounts of IL-1 that are produced by activated monocytes/macrophages as present within blood increase the production of hydrogen peroxide (H2O2) by chondrocytes. The hydrogen peroxide reacts with haemoglobin-derived iron from damaged and phagocytosed red blood cells in the joint cavity, which results in the formation of hydroxyl radicals in the vicinity of chondrocytes. This leads to chondrocyte apoptosis and with that the irreversible inhibition of cartilage matrix synthesis. This primary cartilage-driven joint damage is followed by synovial inflammation adding to joint damage. In search of possible interventions to prevent or limit the cartilage damaging effects of joint bleeds, we evaluated the potential of IL-10. Methods: Healthy human articular cartilage tissue explants were cultured in the presence or absence of 50% v/v blood for 4 days, followed by a recovery period of 12 days. IL-10 was added in 0.1, 1 or 10 ng/ml. Also cartilage and synovium in case of haemophilic arthropathy, obtained at joint replacement surgery, were cultured in the presence of IL-10 (10 ng/ml). The effect on cartilage matrix proteoglycan synthesis, -release, and -content were determined as well as the IL-1β and TNFα production by the synovium. Results: IL-10 was able to prevent dose dependently the decrease in proteoglycan synthesis and the increase in proteoglycan release of cartilage exposed to blood (p<0.05). As a consequence, the decrease in proteoglycan content after blood exposure could be prevented dose dependently (p<0.05). With 10 ng/ml IL-10, which is still a low dose for local therapeutic treatment, at least 50% reduction in adverse effects was observed (p<0.05). The matrix turnover of haemophilic cartilage improved upon addition of IL-10 and also the production of IL-1β and TNFα by haemophilic synovium decreased in the presence of IL-10 (all p<0.05). Conclusions: The present results show that interleukin-10 prevents the direct harmful effects of blood on articular cartilage and has a beneficial effect on the matrix turnover of haemophilic cartilage and pro-inflammatory cytokine production by haemophilic synovium. Therefore, although treatment studies in addition to prevention studies have to be performed; IL-10 might be worthwhile to test in clinical practice for local treatment of joint haemorrhages, to prevent joint damage in time.