Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC-Specific Chimeric Antigen Receptor.

CD4+ CD25high FOXP3+ regulatory T cells (Tregs) are involved in graft-specific tolerance after solid organ transplantation. However, adoptive transfer of polyspecific Tregs alone is insufficient to prevent graft rejection even in rodent models, indicating that graft-specific Tregs are required. We developed a highly specific chimeric antigen receptor that recognizes the HLA molecule A*02 (referred to as A2-CAR). Transduction into natural regulatory T cells (nTregs) changes the specificity of the nTregs without alteration of their regulatory phenotype and epigenetic stability. Activation of nTregs via the A2-CAR induced proliferation and enhanced the suppressor function of modified nTregs. Compared with nTregs, A2-CAR Tregs exhibited superior control of strong allospecific immune responses in vitro and in humanized mouse models. A2-CAR Tregs completely prevented rejection of allogeneic target cells and tissues in immune reconstituted humanized mice in the absence of any immunosuppression. Therefore, these modified cells have great potential for incorporation into clinical trials of Treg-supported weaning after allogeneic transplantation.

[Treatment of lateral ankle joint instability. Open or arthroscopic?]. / Behandlung der lateralen OSG-Instabilität. Offen oder arthroskopisch?

Chronic ankle joint instability often necessitates operative treatment. Operative treatment methods are classified into non-anatomical tenodesis, anatomical reconstruction and direct repair. In addition to open approaches, arthroscopic techniques are increasingly becoming established. This article describes the various operative treatment procedures, their advantages and disadvantages and in particular the arthroscopic feasibility.

Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: implications in cell reprogramming and stem cell therapy.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors.

Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were approximately 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O(6)-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression.

Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

[The chronic central fracture dislocation of the hip]. / Die chronisch fixierte zentrale Hüftgelenkluxationsfraktur.

The acetabulum fracture with chronic dislocation of the femoral head is relatively uncommon. Due to complex morphology and bone defect of the acetabular dome, stable internal fixation is difficult. Primary total hip arthroplasty represents an alternative treatment. We report the case of a 66-year-old patient with a 3-month history of acetabular fracture with central dislocation of the femoral head. The patient was treated with cemented total hip arthroplasty and cancellous bone grafting of the central dome defect for stable cup fixation.

[Arthrolysis for chronic flexion deficits of the knee. An overview of indications and techniques of vastus intermedius muscle resection, transposition of the tibial tuberosity and z-plasty of the patellar tendon]. / Die Arthrolyse bei chronischen Beugedefiziten des Kniegelenks. Eine Ubersicht zu Indikation und Technik von M.-vastus-intermedius-Resektion, Tuberositaskranialisierung und Patellarsehnenverlängerung.

AIM AND METHOD: Limitations to the range of motion of the knee reduce knee function and life quality. Flexion deficits inhibit using stairs and shoe closure, and may cause social deprivation. We present a pathophysiological concept of arthrolysis of the knee for flexion deficit. Our concept divides into intra- and extra-articular factors involving knee stiffness. Extra-articular problems can be located proximally in the quadriceps mechanism and distally in the patellar tendon. RESULTS: The main proximal factor is fibrosis of the vastus intermedius muscle (MVI) which is treated by MVI-resection; the main distal factor is shortening of the patellar tendon which is treated by z-plasty of the this tendon or transposition of the tibial tuberosity. Intra-articular factors are adhesions and fat pad fibrosis. These conditions can be treated by arthroscopic or limited open arthrolysis and eventually z-plasty of the retinacula. CONCLUSION: A strict postoperative protocol is obligatory for pain control and physiotherapy. In 19 cases treated with this algorithm there was a mean flexion gain of 26 degrees. The AOSSM subjective outcome score was excellent in ten and good in seven cases after a mean follow-up of 8.2 months. This treatment protocol allows improvement in flexion, even in difficult revision cases.

[Patella fractures]. / Frakturen der Patella.

Patella fractures are relatively uncommon, accounting for approximately 0.5% to 1.5% of all skeletal injuries. The most common mechanism for this injury is direct fall onto the knee, and transverse fracture is the most common type. The aims of operative treatment are accurate reduction and rigid fixation. Stable fracture types without or with minimal dislocation can be treated nonoperatively. Dislocation of more than 2 mm and comminuted fractures are indications for operative treatment. Tension band wiring, interfragmentary screw fixation, and the combination of cerclage wiring and screw fixation are used for internal fixation of these fractures. When accurate reduction and reconstruction of the retropatellar joint surface cannot be achieved in multi-fragmentary fractures, partial or total patellectomy should be considered. Since it always results in loss of quadriceps muscle power, the decision for this procedure should be made cautiously.

[The minimally-invasive screw osteosynthesis of the medial femoral neck fracture in the very old. A prospective clinical study]. / Die minimal-invasive Schraubenosteosynthese der medialen Schenkelhalsfraktur im höheren Lebensalter. Eine prospektive klinische Studie.

Increasing life expectancy has led to an increase of medial femoral neck fractures. Treatment by hip arthroplasty is a major surgical procedure for geriatric patients and imposes high healthcare costs. Manninger developed a cannulated screw system which allows a joint-sparing, less invasive, and stable internal fixation for the management of intracapsular femoral neck fractures. In a prospective study from January 1998 to August 2002, 63 patients older than 70 years with Garden type I to III fractures were treated by closed reduction and internal fixation with two Manninger screws on a traction table within the first 6 h after trauma. Mean time of follow-up was 21.5+/-16 months. Of the patients examined, 88.5% regained their preoperative stage of mobility. Two patients needed secondary hip arthroplasty. Due to low complication and failure rates and less medical expenses compared to treatment by hemiprosthesis the joint-sparing, less invasive internal fixation with two cannulated Manninger screws presents a safe and cost-efficient surgical technique for geriatric patients.

Simplified MRI sequences for postoperative control of hamstring anterior cruciate ligament reconstruction.

INTRODUCTION: Usually, standard radiographs are used for postoperative quality follow-up after ACL reconstruction. However, with the use of hamstring grafts and bioabsorbable implants, accurate assessment of the tunnel and implant position is impossible. The graft and its relation to anatomical landmarks cannot be evaluated directly. MRI is an alternative to radiography, permitting direct graft visualization and 3-dimensional assessment of the tunnel position, but it is expensive and time consuming for routine use. The aim of this study was to develop a simplified MRI protocol and to evaluate it for routine postoperative quality follow-up after ACL reconstruction. MATERIALS AND METHODS: Various scanning protocols were tested in a series of 105 patients and evaluated for image sharpness, clarity of the structures, susceptibility to artefacts, applicability regarding precise analysis of graft and tunnel position, and time consumption. One simplified specific scan protocol was then defined and applied in a series of 60 consecutive patients after hamstring ACL replacement. The position of the femoral and tibial tunnels was measured in the sagittal, coronal and axial sections and classified according to Harner (femoral) and Stäubli (tibial). Impingement of the graft in the intercondylar roof was analysed according to Howell. The position of the bioabsorbable interference screws was assessed. RESULTS: Scan protocol: T2-weighted gradient-echo sequences (GRE) with TR 246 ms, TE 11 ms, flip angle 25 degrees, 2 mm sections and a 256 x 256 matrix yielded the best image quality of tendon grafts and bone tunnels with tolerable time consumption (average scanning time per patient 1 min 40 s). Altogether 8-16 sections were obtained for every patient. Tunnel placement: 46/60 (77%) of the femoral tunnels were in zone 4, 13/60 (21%) at the border of zones 3 to 4, 1/60 (2%) in zone 3 in the sagittal plane (Harner). The femoral tunnels in the axial plane were at 10:30 o'clock in 32/60 (53%), at 11:00 o'clock at 24/60 (40%) and at 10:00 o'clock in 4/60 (4%) patients. The mean distance of the anterior border of the tibial tunnel from the anterior cortex was 39% (+/- 4.9%) related to the total sagittal diameter of the tibia. There was no graft impingement. The position of the interference screws was anterior to the grafts in all cases. CONCLUSION: Simplified MRI sequences can be used for postoperative quality follow-up after ACL replacement and are an alternative to standard radiographs giving more specific and precise information regarding tunnel position and screw placement. Analyzing the bone tunnels in a series of 60 patients demonstrated that correct assessment of tunnel placement after arthroscopic ACL reconstruction is feasible using this simplified MRI technique.

[The direct, dorsal approach to the treatment of unstable tibial posteromedial fracture-dislocations]. / Der direkte dorsale Zugangsweg zur Versorgung posteromedialer Luxationsfrakturen des Tibiakopfes.

The surgical treatment of highly unstable tibial posteromedial fracture-dislocations (medial split fracture,Moore type I) is demanding and may cause significant problems. The anterior and the posteromedial approach require detachment of the medial capsuloligamental structures or arthrotomy of the knee joint. Neither of these operative techniques allow the optimal posterior positioning of the buttress plate in order to prevent caudal dislocation of the fragment during knee flexion. The published posterior approaches to the proximal tibia allow appropriate positioning of the anti-glide plate. However, these surgical exposures involve transsection of the semi-membranous and popliteal muscles as well as the partial dissection of the medial gastrocnemius muscle. We present a direct posterior approach and an operative treatment of medial split fractures with open reduction and internal fixation in a prone position. This position simplifies the reduction of the posteromedial fragment by hyperextension and axial traction, as well as osteosynthesis with lag screws and the placement of the buttress plate (radial T-plate). The surgical exposure requires minimal soft tissue dissection. The inferior spike of the fragment can be visualized by partial subperiosteal detachment the popliteal muscle,whereas the medial gastrocnemius muscle and the semi-membranous muscle are preserved. Due to the voluminous subcutaneous tissue in the posteromedial tibial aspect, the closure of the skin incision over the medial gastrocnemius muscle can be performed without difficulty. Three cases of tibial posteromedial fracture-dislocation were treated by the direct posterior approach in a prone position in January and February 2002. The present report shows that the direct posterior surgical technique allows optimal exposure, reduction and internal fixation of medial split fractures of the tibial plateau with minimal trauma.

Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.

[Valgus tibial head reconstruction, monocondylar sled prosthesis or bicondylar gliding surface replacement in therapy of medial gonarthrosis--a cost analysis]. / Valgisierende Tibiakopfumstellung, monokondyläre Schlittenprothese oder bikondylärer Gleitflächenersatz zur Therapie der medialen Gonarthrose--eine Kostenstrukturanalyse.

AIM: Three to a certain extent competing methods for the treatment of unicompartmental osteoarthritis of the knee are compared regarding the costs they cause. These methods comprise the high tibial valgus osteotomy (HTO), the unicompartmental knee arthroplasty (UKA), and the total knee arthroplasty (TKA). METHODS: We compared the in-patient costs and out-patient costs of 20 patients (drawn by lot) in each group who received one of the above-named operative methods at our hospital between 1988 and 1993. The results were extrapolated according to the expected survival rate of the applied method. RESULTS: The average total costs of patients who received a HTO were 9.487,-; for the unicompartmental arthroplasty the average cost were 11.687,-; the implantation of a TKA resulted in average costs of 16.940,-. All operative procedures exceeded a particular global amount, the socalled "Fallpauschale". CONCLUSION: Regarding the total costs that arise for the operative treatment of the degenerative arthritis of the knee, the HTO (with or without hardware removal) proves to be the cheapest of the applied methods. Projecting the results on the survival rate of each treatment method, no statistically significant differences could be recognized between the three operative procedures.