Predictive blood coagulation markers for early diagnosis of venous thromboembolism after total knee joint replacement.

Pulmonary embolism development may be prevented if asymptomatic venous thromboembolism (VTE) can be predicted and treated preoperatively or soon after total knee arthroplasty (TKA). The purpose of this study was to evaluate whether asymptomatic VTE can be predicted by blood coagulation markers preoperatively or early after TKA. This prospective single-centre study enrolled 68 patients (6 men, 62 women; mean age: 71 years) who underwent TKA between September 2004 and August 2009. Sixteen-row multidetector computed tomography was performed 4 days before and after surgery for diagnosis of asymptomatic VTE. Blood samples were taken to measure the plasma levels of soluble fibrin monomer complex (SFMC), D-dimer and cross-linked fibrin degradation products by leukocyte elastase (e-XDP) at 4 days preoperatively, and at 1 hour, 1 day and 4 days postoperatively. The preoperative SFMC, D-dimer and e-XDP levels did not differ significantly between the thrombus (n=36) and no-thrombus (n=32) groups. D-dimer and e-XDP levels showed the most significant increases at days 4 and 1, respectively, after surgery in the thrombus group. With cut-off points of 7.5 µg/ml for D-dimer and 8.2 U/ml for e-XDP, the sensitivities were 75% and 75%, and the specificities were 63% and 59%, respectively. By multiple logistic regression analysis, D-dimer at day 4 and e-XDP at day 1 postoperatively were independent markers for early diagnosis of VTE (odds ratio=1.61 and 1.19, P=0.01 and 0.04, respectively). The postoperative occurrence of new asymptomatic VTE may be predicted by D-dimer at day 4 and e-XDP at day 1 after TKA.

The incidence of venous thromboembolism before and after total knee arthroplasty using 16-row multidetector computed tomography.

We performed a prospective study to determine the incidence of venous thromboembolism (VTE) using 16-row multidetector computed tomography (MDCT). The study included 71 patients who underwent total knee arthroplasty between September 2004 and March 2009. Multidetector computed tomography was performed 4 days before and after surgery. No patient had any presurgical symptoms of VTE. Presurgical and postsurgical incidences of pulmonary thromboembolism plus deep vein thrombosis were 0% and 13%, respectively; pulmonary thromboembolism alone, 1% and 3%, respectively; and deep vein thrombosis alone, 8% and 34%, respectively. Because asymptomatic VTE was noted in 9% of patients before surgery and 51% after surgery, we conclude that performing MDCT before and after total knee arthroplasty may be useful to clarify the incidence of VTE and to develop appropriate strategies for treatment and prevention.

Arthroscopic-assisted tibiotalocalcaneal arthrodesis using an intramedullary nail with fins: a case report.

Arthroscopic arthrodesis of the ankle has become popular because of the reduced invasiveness of the procedure and good bony consolidation compared with conventional open techniques. However, arthroscopic arthrodesis of the subtalar joint has not been as universally accepted. Rheumatoid arthritis frequently involves the talocalcaneal joint in addition to the tibiotalar joint. In such cases, simultaneous fixation of both tibiotalar and talocalcaneal joints is desirable. We undertook arthroscopic-assisted arthrodesis of the tibiotalocalcaneal joint using intramedullary nails with fins for a 76-year-old man with rheumatoid arthritis. Although the patient presented with poor skin condition and osteoporotic bone due to long-term use of systemic corticosteroids, weight bearing was allowed 2 weeks after the surgery. Solid fusion of the tibiotalocalcaneal joint occurred without any complications. Given the twin benefits of reduced invasiveness and secure fixation, this method should be considered for patients requiring both tibiotalar and talocalcaneal joint fusion, when a more extensive surgical exposure would be more risky.

Histochemical study on the changes in muscle fibers in relation to the effects of aging on recovery from muscular atrophy caused by disuse in rats.

To investigate the effects of aging on the degree of muscular atrophy caused by disuse and its recovery, we evaluated the recovery from muscular atrophy induced in both young and old rats under the same conditions. The soleus was atrophied by immobilization of the foot joint in a hindlimb and unweighting of the bilateral hindlimbs for 2 weeks, and measurement of the wet weight of muscles and biochemical examination were performed 2, 4, and 6 weeks after the removal of unweighting and fixation during the recovery period of 6 weeks. There was no difference in the degree of atrophy in the fixed soleus between the young and old rats. The recovery from atrophy was delayed in the older rats compared to the young rats. In the unfixed hindlimb, the degree of atrophy was low in both the old and young rats, and the recovery was rapid. Because the recovery from disuse muscular atrophy is delayed with aging, it is necessary to avoid unweighting and immobilization, or to reduce the period spent under such conditions.

Immunohistochemical study of the proliferation modality of synovium in rat adjuvant arthritis.

To investigate the proliferative potencies of the two types of synovial lining cells (types A and B), we used immunohistochemical techniques under light and electron microscopy to survey the complete process of arthritis in a rat model system. Complete Freund's adjuvant (0.1 ml) containing 5 mg of heat-inactivated Mycobacterium butyricum was administered intradermally into the right hind paws of 25 male Sprague-Dawley rats. Five animals were killed at weeks 1, 2, 3, and 4 after the immunological challenge, respectively, and the infrapatellar adipose synovium was removed from each animal. Using proliferative cell nuclear antigen (PCNA) as a cell proliferation marker, we quantified the locations of proliferative cells under light microscopy and then identified the cell type with immunoelectron microscopy. The number of PCNA-positive cells was high in the deep layer at week 2, and most of them were determined to be B cells. At week 3, the number of PCNA-positive cells increased in the superficial layer, and most were identified as A cells. Our results support the idea that cells of the synovial lining proliferate in situ during the course of arthritis.

Long-lasting gene expression by particle-mediated intramuscular transfection modified with bupivacaine: combinatorial gene therapy with IL-12 and IL-18 cDNA against rat sarcoma at a distant site.

The immune response is modulated by genetic adjuvants using plasmid vectors expressing cytokines. Skeletal muscle can express a foreign gene intramuscularly administered via a needle injection, and the potential of muscle as a target tissue for somatic gene therapy in treating cancer has been explored. In the present study, we investigated the efficacy of particle-mediated intramuscular transfection modified with a local anesthetic agent, bupivacaine, on luciferase and green fluorescent protein. The results indicate that these proteins are more efficiently expressed and persist longer in muscle modified in this way compared with the needle-injection method. Using an established rat sarcoma model, particle-mediated intramuscular gene-gun therapy with a combination of IL-12 and IL-18 cDNA was conducted. Growth of the distant sarcoma was significantly inhibited by particle-mediated intramuscular combination gene therapy, and the survival rate was also improved. Furthermore, the combination gene-gun therapy maintained significant levels of interferon-gamma and induced a high activity of tumor-specific cytotoxic T lymphocytes. These results suggest that the sustained local delivery of IL-12 and IL-18 cDNA using intramuscular gene-gun therapy modified with bupivacaine can induce long-term antitumor immunity, and can provide the great advantage of inhibiting the disseminated tumor.

Defective sorting to secretory vesicles in trans-Golgi network is partly responsible for protein C deficiency: molecular mechanisms of impaired secretion of abnormal protein C R169W, R352W, and G376D.

Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Generation of donor hematolymphoid cells after rat-limb composite grafting.

BACKGROUND: Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. METHODS: Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP donor onto the GFP recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. RESULTS: The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. CONCLUSIONS: The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.