CD99 triggering in Ewing sarcoma delivers a lethal signal through p53 pathway reactivation and cooperates with doxorubicin.

PURPOSE: The paucity of new drugs for the treatment of Ewing sarcoma (EWS) limits the cure of these patients. CD99 has a strong membranous expression in EWS cells and, being also necessary for tumor survival, is a suitable target to aim at. In this article, we described a novel human monospecific bivalent single-chain fragment variable diabody (dAbd C7) directed against CD99 of potential clinical application. EXPERIMENTAL DESIGN: In vitro and in vivo evaluation of cell death and of the molecular mechanisms triggered by anti-CD99 agents were performed alone or in combination with doxorubicin to demonstrate efficacy and selectivity of the new dAbd C7. RESULTS: The dAbd C7 induced rapid and massive EWS cell death through Mdm2 degradation and p53 reactivation. Mdm2 overexpression as well as silencing of p53 in p53wt EWS cells decreased CD99-induced EWS cell death, whereas treatment with nutlin-3 enhanced it. Furthermore, cell death was associated with induction of p21, bax, and mitochondrial depolarization together with substantial inhibition of tumor cell proliferation. Combined treatment of anti-CD99 dAbd C7 with doxorubicin was additive both in vitro and in vivo against EWS xenografts. Normal mesenchymal stem cells showed no p53 activation and were resistant to cell death, unless transformed by EWS-FLI, the oncogenic driver of EWS. CONCLUSIONS: These results indicate that dAbd C7 is a suitable candidate tool to target CD99 in patients with EWS able to spare normal stem cells from death as it needs an aberrant genetic context for the efficient delivery of CD99-triggered cell death.

Reconstruction with fascia lata after extensive chest wall resection: results.

OBJECTIVES: Following extensive chest wall resection, the reconstruction technique should fulfill two opposing functional requirements: adequate rigidity and flexibility of the chest wall during the breathing phases. Reconstruction with fascia lata enables a balance between these two parameters, thus favouring the patients' respiratory dynamics and producing low morbidity and good functional results. METHODS: Sixty patients underwent chest wall reconstruction using fascia lata alone or in combination with titanium plates between 2006 and 2011, due to primary tumours in 28 patients, metastases in 23 and local recurrences in 9. The mean area of resected tissue was 107.7 cm(2), distributed among the anterior, anterolateral, lateral and posterior zones. One-to-eight ribs were resected, and additional sternum resection was performed in 75% of patients. Forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were evaluated in 33 patients. RESULTS: 46.6% of patients underwent reconstruction with fascia lata alone, 1.6% with fascia lata, DualMesh® and titanium plates and 51.6% with fascia lata and titanium plates. There was no 30-day mortality. All patients were extubated after the operation with no need for reintubation. Five patients had postoperative complications: 2 wound dehiscences, 2 haematomas and 1 seroma. There were no significant differences between preoperative and postoperative FEV1 and FVC measurements in patients with or without lobectomy and wedge resections. CONCLUSIONS: Chest wall reconstruction with fascia lata, alone or in combination with titanium plates, allows the surgeon to perform a dynamic reconstruction without flail chest in extensive exeresis. Risks of infection associated with the use of prosthetic materials are also minimized. In addition, the characteristic flexibility of this tissue makes it a precious tool in paediatric chest wall reconstruction, since fascia lata naturally adapts to the physiological growth of younger bodies, thus reducing the risk of scoliosis and local deformities.

Human mesenchymal stem cells: a bank perspective on the isolation, characterization and potential of alternative sources for the regeneration of musculoskeletal tissues.

The continuous discovery of human mesenchymal stem cells (hMSCs) in different tissues is stirring up a tremendous interest as a cell source for regenerative medicine therapies. Historically, hMSCs have been always considered a sub-population of mononuclear cells present in the bone marrow (BM). Although BM-hMSCs are still nowadays considered as the most promising mesenchymal stem cell population to reach the clinics due to their capacity to differentiate into multiple tissues, hMSCs derived from other adult and fetal tissues have also demonstrated to possess similar differentiation capacities. Furthermore, different reports have highlighted a higher recurrence of hMSCs in some of these tissues as compared to BM. This offer a fascinating panorama for cell banking, since the creation of a stem cell factory could be envisioned where hMSCs are stocked and used for ad hoc clinical applications. In this review, we summarize the main findings and state of the art in hMSCs isolation, characterization, and differentiation from alternative tissue sources and we attempt to compare their potency for musculoskeletal regeneration.

Platelet-rich plasma vs hyaluronic acid to treat knee degenerative pathology: study design and preliminary results of a randomized controlled trial.

BACKGROUND: Platelet rich plasma (PRP), a blood-derived product rich in growth factors, is a promising treatment for cartilage defects but there is still a lack of clinical evidence. The aim of this study is to show, through a randomized double blind prospective trial, the efficacy of this procedure, by comparing PRP to Hyaluronic Acid (HA) injections for the treatment of knee chondropathy or osteoarthritis (OA). METHODS: 109 patients (55 treated with HA and 54 with PRP) were treated and evaluated at 12 months of follow-up. The patients were enrolled according to the following inclusion criteria: age > 18 years, history of chronic (at least 4 months) pain or swelling of the knee and imaging findings of degenerative changes of the joint (Kellgren-Lawrence Score up to 3). A cycle of 3 weekly injections was administered blindly. All patients were prospectively evaluated before and at 2, 6, and 12 months after the treatment by: IKDC, EQ-VAS, TEGNER, and KOOS scores. Range of motion and knee circumference changes were measured over time. Adverse events and patient satisfaction were also recorded. RESULTS: Only minor adverse events were detected in some patients, such as mild pain and effusion after the injections, in particular in the PRP group, where a significantly higher post-injective pain reaction was observed (p=0.039). At the follow-up evaluations, both groups presented a clinical improvement but the comparison between the two groups showed a not statistically significant difference in all scores evaluated. A trend favorable for the PRP group was only found in patients with low grade articular degeneration (Kellgren-Lawrence score up to 2). CONCLUSIONS: Results suggest that PRP injections offer a significant clinical improvement up to one year of follow-up. However, conversely to what was shown by the current literature, for middle-aged patients with moderate signs of OA, PRP results were not better than those obtained with HA injections, and thus it should not be considered as first line treatment. More promising results are shown for its use in low grade degeneration, but they still have to be confirmed.

Bipolar fresh osteochondral allograft for the treatment of glenohumeral post-traumatic arthritis.

PURPOSE: Surgical management of end-stage glenohumeral osteoarthritis represents a challenge in young patients. Bipolar fresh osteochondral allograft (BFOA) represents a fascinating option to prosthetic substitution. Aim of this report is to describe an original technique for BFOA implantation in the shoulder and to describe the clinical and histological results at 30-month follow-up. METHODS: A 47-year-old man received BFOA of the shoulder. Clinical and radiographical evaluations were carried out periodically up to 30 months. RESULTS: The clinical score increased from 40 to 78 points (Constant Score), decreased from 64 points to 10 (DASH Disability/Symptom Score) and from 94 points to 0 (DASH Work module Score). Radiographically arthritis and partial reabsorption of the implanted surfaces were evident. CONCLUSIONS: The clinical result obtained in this patient seems to support the applicability of BFOA in the shoulder with severe post-traumatic arthritis and with intact rotator cuff. Although unrelated to the clinical result, the occurrence of arthritis of the implanted surfaces is cause of concern. LEVEL OF EVIDENCE: Case report, Level V.

Platelet-rich plasma intra-articular injections for cartilage degeneration and osteoarthritis: single- versus double-spinning approach.

PURPOSE: To compare the safety and efficacy of two different approaches of platelet-rich plasma (PRP) production methods as intra-articular injection treatment for knee cartilage degenerative lesions and osteoarthritis (OA). METHODS: The study involved 144 symptomatic patients affected by cartilage degenerative lesions and OA. Seventy-two patients were treated with 3 injections of platelet concentrate prepared with a single-spinning procedure (PRGF), the other 72 with 3 injections of PRP obtained with a double-spinning approach. The patients were evaluated prospectively at the enrollment and at 2, 6, and 12 months' follow-up with IKDC, EQ-VAS and Tegner scores; adverse events and patient satisfaction were also recorded. RESULTS: Both treatment groups presented a statistically significant improvement in all the scores evaluated at all the follow-up times. Better results were achieved in both groups in younger patients with a lower degree of cartilage degeneration. The comparative analysis showed similar improvements with the two procedures: in particular, IKDC subjective evaluation increased from 45.0 ± 10.1 to 59.0 ± 16.2, 61.3 ± 16.3, and 61.6 ± 16.2 at 2, 6, and 12 months in the PRGF group, and from 42.1 ± 13.5 to 60.8 ± 16.6, 62.5 ± 19.9, and 59.9 ± 20.0 at 2, 6, and 12 months in the PRP group, respectively. Concerning adverse events, more swelling (P = 0.03) and pain reaction (P = 0.0005), were found after PRP injections. CONCLUSIONS: Although PRP injections produced more pain and swelling reaction with respect to that produced by PRGF, similar results were found at the follow-up times, with a significant clinical improvement with respect to the basal level. Better results were achieved in younger patients with a low degree of cartilage degeneration. LEVEL OF EVIDENCE: II.

Platelet-rich plasma intra-articular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis.

PURPOSE: The aim of our study is to compare the efficacy of platelet-rich plasma (PRP) and viscosupplementation (hyaluronic acid [HA]) intra-articular injections for the treatment of knee cartilage degenerative lesions and osteoarthritis (OA). METHODS: The study involved 150 patients affected by cartilage degenerative lesions and early and severe OA. Fifty symptomatic patients were treated with 3 autologous PRP intra-articular injections and were evaluated prospectively at enrollment and at 2- and 6-month follow-up. The results obtained were compared with 2 homogeneous groups of patients treated with HA injections. One group was treated with injections of high-molecular weight HA; the other group was treated with low-molecular weight (LW) HA. International Knee Documentation Committee and EQ VAS scores were used for clinical evaluation; adverse events and patient satisfaction were also recorded. RESULTS: At 2 months' follow-up, the PRP and LW HA groups showed a similar improvement, with higher results compared with the high-molecular weight HA group (P < .005). At 6 months' follow-up, better results were observed in the PRP group (P < .005). PRP and LW HA treatments offered similar results in patients aged over 50 years and in the treatment of advanced OA. PRP showed a better performance compared with HA in younger patients affected by cartilage lesions or early OA. CONCLUSIONS: Autologous PRP injections showed more and longer efficacy than HA injections in reducing pain and symptoms and recovering articular function. Better results were achieved in younger and more active patients with a low degree of cartilage degeneration, whereas a worse outcome was obtained in more degenerated joints and in older patients, in whom results similar to those of viscosupplementation have been observed. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Comparison of alternative mesenchymal stem cell sources for cell banking and musculoskeletal advanced therapies.

With the continuous discovery of new alternative sources containing mesenchymal stem cells (MSCs), regenerative medicine therapies may find tailored applications in the clinics. Although these cells have been demonstrated to express specific mesenchymal markers and are able to differentiate into mesenchymal lineages in ad hoc culture conditions, it is still critical to determine the yield and differentiation potential of these cells in comparative studies under the same standardized culture environment. Moreover, the opportunity to use MSCs from bone marrow (BM) of multiorgan donors for cell banking is of relevant importance. In the attempt to establish the relative potential of alternative MSCs sources, we analyzed and compared the yield and differentiation potential of human MSCs from adipose and BM tissues of cadaveric origins, and from fetal annexes (placenta and umbilical cord) after delivery using standardized isolation and culture protocols. BM contained a significantly higher amount of mononuclear cells (MNCs) compared to the other tissue sources. Nonetheless, a higher cell seeding density was needed for these cells to successfully isolate MSCs. The MNCs populations were highly heterogeneous and expressed variable MSCs markers with a large variation from donor to donor. After MSCs selection through tissue culture plastic adhesion, cells displayed a comparable proliferation capacity with distinct colony morphologies and were positive for a pool of typical MSCs markers. In vitro differentiation assays showed a higher osteogenic differentiation capacity of adipose tissue and BM MSCs, and a higher chondrogenic differentiation capacity of BM MSCs.

Platelet-rich plasma intra-articular knee injections for the treatment of degenerative cartilage lesions and osteoarthritis.

PURPOSE: Platelet-rich plasma (PRP) therapy is a simple, low-cost and minimally invasive method that provides a natural concentrate of autologous blood growth factors (GFs) that can be used to enhance tissue regeneration. In a previous analysis of a 12-month follow-up study, promising results were obtained when treating patients affected by knee degeneration with PRP intra-articular injections. The main purpose of this study was to investigate the persistence of the beneficial effects observed. METHODS: Of the 91 patients evaluated in the previous 12-month follow-up study, 90 were available for the 2-year follow-up (24 patients presented a bilateral lesion, in a total of 114 knees treated). All of the patients presented a chronic knee degenerative condition and were treated with three intra-articular PRP injections. IKDC and EQ-VAS scores were used for clinical evaluation. Complications, adverse events and patient satisfaction were also recorded. RESULTS: All of the evaluated parameters worsened at the 24-month follow-up: these parameters were at significantly lower levels with respect to the 12-month evaluation (the IKDC objective evaluation fell from 67 to 59% of normal and nearly normal knees; the IKDC subjective score fell from 60 to 51), even if they remained higher than the basal level. Further analysis showed better results in younger patients (P = 0.0001) and lower degrees of cartilage degeneration (P < 0.0005). The median duration of the clinical improvement was 9 months. CONCLUSIONS: These findings indicate that treatment with PRP injections can reduce pain and improve knee function and quality of life with short-term efficacy. Further studies are needed to confirm these results and understand the mechanism of action, and to find other application modalities, with different platelet and GF concentrations and injection timing, which provide better and more durable results.

Platelet-rich plasma: intra-articular knee injections produced favorable results on degenerative cartilage lesions.

Platelet-rich plasma (PRP) is a natural concentrate of autologous blood growth factors experimented in different fields of medicine in order to test its potential to enhance tissue regeneration. The aim of our study is to explore this novel approach to treat degenerative lesions of articular cartilage of the knee. One hundred consecutive patients, affected by chronic degenerative condition of the knee, were treated with PRP intra-articular injections (115 knees treated). The procedure consisted of 150-ml of venous blood collected and twice centrifugated: 3 PRP units of 5 ml each were used for the injections. Patients were clinically prospectively evaluated before and at the end of the treatment, and at 6 and 12 months follow-up. IKDC, objective and subjective, and EQ VAS were used for clinical evaluation. Statistical analysis was performed to evaluate the significance of sex, age, grade of OA and BMI. A statistically significant improvement of all clinical scores was obtained from the basal evaluation to the end of the therapy and at 6-12 months follow-up (P < 0.0005). The results remained stable from the end of the therapy to 6 months follow up, whereas they became significantly worse at 12 months follow up (P = 0.02), even if still significantly higher respect to the basal level (P < 0.0005). The preliminary results indicate that the treatment with PRP injections is safe and has the potential to reduce pain and improve knee function and quality of live in younger patients with low degree of articular degeneration.

Use of platelet-rich plasma for the treatment of refractory jumper's knee.

The aim of this study was to evaluate the efficacy of multiple platelet-rich plasma (PRP) injections on the healing of chronic refractory patellar tendinopathy after previous classical treatments have failed. We treated 15 patients affected by chronic jumper's knee, who had failed previous nonsurgical or surgical treatments, with multiple PRP injections and physiotherapy. We also compared the clinical outcome with a homogeneous group of 16 patients primarily treated exclusively with the physiotherapy approach. Multiple PRP injections were performed on three occasions two weeks apart into the site of patellar tendinopathy. Tegner, EQ VAS and pain level were used for clinical evaluation before, at the end of the treatment and at six months follow-up. Complications, functional recovery and patient satisfaction were also recorded. A statistically significant improvement in all scores was observed at the end of the PRP injections in patients with chronic refractory patellar tendinopathy and a further improvement was noted at six months, after physiotherapy was added. Moreover, comparable results were obtained with respect to the less severe cases in the EQ VAS score and pain level evaluation, as in time to recover and patient satisfaction, with an even higher improvement in the sport activity level achieved in the PRP group. The clinical results are encouraging, indicating that PRP injections have the potential to promote the achievement of a satisfactory clinical outcome, even in difficult cases with chronic refractory tendinopathy after previous classical treatments have failed.

Platelet-rich plasma: new clinical application: a pilot study for treatment of jumper's knee.

This study describes a simple, low-cost, minimally invasive way to apply PRP growth factors to chronic patellar tendinosis; 20 male athletes with a mean history of 20.7 months of pain received treatment, and outcomes were prospectively evaluated at 6 months follow-up. No severe adverse events were observed, and statistically significant improvements in all scores were recorded. The results suggest that this method may be safely used for the treatment of jumper's knee, by aiding the regeneration of tissue which otherwise has low healing potential.

In vitro evaluation of freeze-dried bone allografts combined with platelet rich plasma and human bone marrow stromal cells for tissue engineering.

Freeze-dried bone allograft (FDBA) might be more effective in combination with platelet rich plasma (PRP) and bone marrow stromal cells (BMSC) in accelerating bone healing. The isolation of BMSC through density gradient (pBMSC) is not extensively applicable in clinical practice, because it increases the risk of infection. Alternatively, BMSC can be concentrated by simple centrifugation (wBMSC) directly in the operating room. However, we do not know if wBMSC act in the same way as pBMSC. BMSC from 10 donors were tested whether, in the presence of a combination of FDBA and autologous PRP, the osteogenic differentiation of the cells concentrated by simple centrifugation (wBMSC + FDBA + PRP) was similar to that of pBMSC. Cell-associated alkaline phosphatase, osterix and fibroblast growth factor-2 were higher in wBMSC + FDBA + PRP. In conclusion, the combination of FDBA and PRP had a favouring effect on the differentiation towards osteoblasts and allowed BMSC concentrated by simple centrifugation to differentiate as fast as BMSC purified by density gradient.

Cell manipulation in autologous chondrocyte implantation: from research to cleanroom.

In the field of orthopaedics, autologous chondrocyte implantation is a technique currently used for the regeneration of damaged articular cartilage. There is evidence of the neo-formation of tissue displaying characteristics similar to hyaline cartilage. In vitro chondrocyte manipulation is a crucial phase of this therapeutic treatment consisting of different steps: cell isolation from a cartilage biopsy, expansion in monolayer culture and growth onto a three-dimensional biomaterial to implant in the damaged area. To minimise the risk of in vitro cell contamination, the manipulation must be performed in a controlled environment such as a cleanroom. Moreover, the choice of reagents and raw material suitable for clinical use in humans and the translation of research protocols into standardised production processes are important. In this study we describe the preliminary results obtained by the development of chondrocyte manipulation protocols (isolation and monolayer expansion) in cleanrooms for the application of autologous implantation.

Radiological assessment of bone segments for transplantation: experience at Rizzoli Orthopedic Institute.

AIM: To analyse results obtained from radiological assessment of skeletal segments stored in the musculoskeletal tissue bank (MTB) at Rizzoli Orthopaedic Institute. MATERIALS AND METHODS: Between January 1997 and June 2003, 891 bone segments underwent radiographic examination in two views. Two hundred and thirteen of these segments were examined by a radiologist between July 2002 and June 2003. Diagnostic evaluation was aimed at recognising relevant degenerative, traumatic and focal lesions. Focal lesions underwent histological tests. RESULTS: Twenty-two lytic lesions were found in 12 segments. Ten of these were studied between July 2002 and June 2003 and two in the period before specialist radiological assessment. In the latter cases the lesions were identified by the orthopedic specialist who had examined the X-rays before planning surgery. Histological tests showed that the bone tissue was normal or involved by degenerative phenomena. CONCLUSIONS: We think donor screening should include radiological assessment of bone segments, performed according to standard parameters by a radiologist to identify bone lesions that may jeopardise the successful outcome of surgery.

Chelation therapy and bone metabolism markers in thalassemia major.

The aim of our study was to investigate the effects of subcutaneous desferrioxamine (DFX) and oral deferiprone (L1) therapy on bone metabolism markers in patients with thalassemia major. We studied 17 patients with thalassemia receiving long-term treatment with desferrioxamine, 20 patients receiving long-term treatment with deferiprone, and 15 healthy age-matched controls. The following investigations were performed: a) intact parathyroid hormone (PTH), 25-hydroxyvitamin D [25(OH)D], 1,25-dihydroxyvitamin D [1,25(OH)2D] as endocrine parameters; b) alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), osteocalcin (OC); c) bone resorption biochemical markers in serum and urine pyridinium crosslinks: hydroxylysyl-pyridinoline (HP) and lysyl-pyridinoline (LP); d) serum levels of cytokines and growth factors: transforming growth factor-beta1 (TGFbeta1), insulin-like growth factor-I (IGF-I), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-a (TNFalpha); e) serum levels of IGF binding protein-3 (IGFBP-3). No significant differences among all studied variables were found in patients with thalassemia treated with desferrioxamine or deferiprone. In contrast, significant differences were found between patients with thalassemia and the control group: intact PTH was significantly lower in patients with thalassemia than in the controls (p < 0.0005), and a significant increase in ALP and BALP (p < 0.0005), but not in OC, was found in the patient group. With regard to bone resorption and remodeling markers, the urinary excretion of pyridinium crosslinks was higher in patients with thalassemia for HP fraction (p < 0.0005) and LP fraction (p = 0.002), as well as TGFbeta (p = 0.001). In contrast, IGF-I and IGFBP-3 were reduced when compared with controls. In conclusion, the study of bone metabolism markers in adult patients with thalassemia reveals a complex behavior with an increase in bone resorption indexes. Bone formation did not appear to be impaired. In particular, TGFbeta1 was higher in patients with thalassemia receiving L1 treatment.

Stromal stem cells and platelet-rich plasma improve bone allograft integration.

Early vascular invasion is a key factor in bone allograft incorporation. It may reduce the complications related to slow and incomplete bone integration. Bone-marrow-derived stromal stem cells associated with platelet-rich plasma are potent angiogenic inducers proven to release vascular endothelial growth factor. Our goal was to test whether the combination of stromal stem cells and platelet-rich plasma is able to increase massive allograft integration in a large animal model with sacrifice at 4 months. A critical defect was made in the mid-diaphysis of the metatarsal bone of 10 sheep; the study group received an allograft plus stromal stem cells, platelet-rich plasma, and collagen (six animals) and the control group received only the allograft (four animals). Investigation was done with radiographs, mechanical tests and histomorphometric analysis, including new vascularization. Results showed substantial new bone formation in the allograft of the study group. Bone formation is correlated with better vascular invasion and remodeling of the graft in the study group. These results confirm the key role played by stromal stem cells and platelet-rich plasma in bone repair. Further studies are needed to better define the role stromal stem cells play when implanted alone.

Bone reconstruction of large defects using bone marrow derived autologous stem cells.

Bone is a tissue that has the ability to heal itself when fractured. Occasionally, a critical defect can be formed when part of the bone is lost or excised, in this case the bone fails to heal and requires bone reconstruction to prevent a non-union defect. Autogenous cancellous bone is the current gold standard treatment in bone loss. Because the amount of autogenous cancellous bone that can be harvested is limited, the expanding need for bone reconstruction is paired by the growth of interest in the discipline of tissue engineering. Labs worldwide are working to provide the right carrier and the right set of cells that, once retransplanted, will ensure bone repair. Several investigators have focused their attention on a subset of autologous non-hematopoietic stem/progenitor cells contained in the adult bone marrow stroma, referred to as stromal stem cells (SSC), as the appropriate cells to be transplanted. The use of autologous cells is facilitated by less stringent ethical and regulatory issues and does not require the patient to be immunologically suppressed. In pre-clinical and clinical protocols of critical defects in which SSC are employed, two approaches are mainly used: in the first, SSC are derived from bone marrow and directly introduced at the lesion site, in the second, SSC are derived from several sites and are expanded ex vivo before being implanted. Both approaches, equally correct in principle, will have to demonstrate, with definitive evidence of their efficacy, their capability of solving a critical clinical problem such as non-union. In this report we outline the difficulties of working with SSC.

Lymphocyte DNA damage precedes DNA repair or cell death after orthopaedic surgery under general anaesthesia.

Anaesthetics have gained a lot of attention for their potential mutagenic/carcinogenic effects. In the present study we have investigated the genotoxicity of the inhalation anaesthetic sevoflurane on DNA of lymphocytes isolated from 20 patients undergoing orthopaedic surgery. The genotoxicity of the anaesthetic was studied by assaying DNA damage, apoptosis, DNA repair enzyme activity and GSH content in peripheral lymphocytes before, 15 min after anaesthesia and 24 h after surgery. Lymphocytes isolated 15 min after anaesthesia showed an increase in oxidized purine and pyrimidine bases without DNA strand break formation. DNA strand breaks occurred on the first post-operative day, associated with an enhancement of DNA repair activity and a decrease in GSH. Formation of strand breaks could be the consequence of DNA repair activity. In fact, at 24 h after surgery most of the oxidized DNA bases were repaired. When DNA damage was not repaired, activation of the cell cycle checkpoint protein p53 could lead to apoptosis. An altered redox status may contribute to lymphocytopenia due to an apoptotic event as a consequence of surgical trauma. The presence of apoptotic cells at 1 day after surgery could support the hypothesis that highly damaged peripheral lymphocytes are committed to undergo programmed cell death if the damage is not repaired. In conclusion, the actual risk from anaesthesia is presumably extremely small. However, these findings contribute to our understanding of the regulation of DNA damage/repair and cell death.