Ingo H. Pilz

High-resolution insertion-site analysis by linear amplification–mediated PCR (LAM-PCR)

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology—linear amplification–mediated (LAM) PCR—for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette–specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5′ long terminal repeat (LTR) retroviral vector adjacent genomic sequences (Fig. 1 and Box 1).

Expression of BMP-receptor type 1A correlates with progress of osteoarthritis in human knee joints with focal cartilage lesions

BACKGROUND AIMS Bone morphogenetic protein-2 (BMP-2) and its receptor type 1A (BMPR-1A) play significant roles in cartilage metabolism. The aim of this study was to evaluate a possible correlation between intra-articular expression of these proteins and the degree of osteoarthritis (OA) in human knees. METHODS Biopsies of synovia and debrided cartilage were taken in 15 patients undergoing autologous chondrocyte implantation. Expression of BMP-2 and BMPR-1A was evaluated semi-quantitatively by immunohistologic staining. These data were complemented by grading of cartilage lesions according to International Cartilage Repair Society (ICRS), defect size, duration of complaints, knee osteoarthritis scoring system (KOSS) and Henderson and Kellgren-Lawrence scores. General histologic stainings were used to determine Mankin, Pritzker and Krenn scores. RESULTS The expression of BMPR-1A but not of BMP-2 was significantly higher in cartilage biopsies taken in type 3 lesions with intact subchondral layer compared with type 4 defects (P < 0.05). In cartilage areas of bordering sectors, the intensity of immunohistologic staining of BMPR-1A was statistically significantly higher in mature cartilage compared with repair zones (P < 0.05). Expression of BMP-2 and its receptor 1A correlated in the cartilage biopsies (P < 0.02) but not in the synovia. The degree of OA was scored in all biopsies according to Mankin and Pritzker, and these scores correlated statistically significantly with BMPR-1A expression in the synovia (P < 0.05). In patients with an osteochondritis dissecans, the degree of OA was higher compared with other causes of chondromalacia, as evaluated by defect size, ICRS score, duration of complaints, Pritzker score and expression of BMPR-1A in cartilage (P < 0.05). CONCLUSIONS These data support the role of BMPR-1A as an indicator of OA progression in human knees with circumscribed cartilage lesions.

Immunohistological Localization of BMP-2, BMP-7, and Their Receptors in Knee Joints with Focal Cartilage Lesions

Introduction. Although it is well known that BMP-2 and BMP-7 play significant roles in cartilage metabolism, data about intra-articular expression and localization of these proteins and their receptors in humans are rare. Methods. Biopsies of synovia and debrided cartilage were taken in patients undergoing autologous chondrocyte implantation. Expression of BMP-2, BMP-7, and their receptors BMPR-1A, BMPR-1B and BMPR-2 were semiquantitatively evaluated by immunohistological staining. Results. BMP-7 was equally highly expressed in all cartilage and synovial biopsies. Increased levels of BMPR-1A, but not of BMPR-1B, and BMPR-2, were found in all synovial and 47% of all cartilage samples (P = 0.002). BMP-2 was positively scored in 47% of all cartilage and 40% of all synovial specimens. Defect size, KOSS, Henderson or Kellgren-Lawrence score did not statistically significant correlate with the expression of the analyzed proteins or Mankin and Pritzker scores. Duration of symptoms and localization of lesions were associated with KOSS (P < 0.02), but there was no influence of these parameters on protein expression. Conclusions. BMP-2, BMP-7, and BMPR-1A were expressed in cartilage and synovia of knees with focal cartilage lesions. Although defect localization and duration of symptoms decisively influence KOSS, there was no associated alteration of protein expression observed.

Prospective clinical trial of patients who underwent ankle arthroscopy with articular diseases to match clinical and radiological scores with intra-articular cytokines

ObjectiveThere is still a lack of reliable data on cytokine concentrations in the ankle and their value for prognosis.MethodsIn a prospective clinical trial, lavage fluids were collected from 49 patients with an arthroscopy of the ankle. The fluids were investigated by ELISA for cytokine levels. Clinical scores (FFI, AOFAS) were evaluated both pre-operatively and then again 12 months after surgery (n = 43, 88%). Radiological changes were noted with the Kellgren-Lawrence-Score (KLS) and the Ankle Osteoarthritis Scoring System (AOSS). Based on the difference between the pre- and postoperative clinical scores, two groups were defined according to whether they had benefited from the surgical therapy (Δ score ≥ 10) or not (Δ score < 10).ResultsThe average clinical scores had improved to a statistically significant extent in the one-year follow-up (p < 0.01). BMP-2 (p = 0.02), IGF-1 (p = 0.04), BMP-7 (p = 0.01) and aggrecan (p = 0.04) showed significant correlations with pre-operative clinical and radiological scores (p = 0.02, p = 0.01, p = 0.01, p = 0.01). Furthermore, BMP-2 (p = 0.01), IGF-1/TPC (p = 0.03) and aggrecan (p = 0.01) correlated with scores after one year (p = 0.02, p = 0.01). High aggrecan concentrations were associated with a low clinical and a high radiological score at both time points, both indicating progress of cartilage degeneration in contrast to BMP-2 or IGF-1. Furthermore, MMP-13 concentrations were significantly higher in the non-benefit group (p = 0.02).ConclusionBMP-2, IGF-1, aggrecan and MMP-13 seem to be involved in the degenerative process of cartilage in the ankle joint. Additionally, high synovial MMP-13 concentrations indicate a worse clinical outcome.