Plaque-infiltrating T lymphocytes in patients with carotid atherosclerosis: an insight into the cellular mechanisms associated to plaque destabilization.

AIM: Inflammation plays a crucial role in the progression of atherosclerotic plaques. The aim of the present study was to investigate phenotypic and functional characteristics of plaque-infiltrating T lymphocytes associated with a complicated phenotype of carotid atherosclerotic lesions. METHODS: Atherosclerotic plaques were obtained from 17 patients undergoing carotid endarterectomy and cultured to isolate infiltrating T lymphocytes. Blood samples were obtained from patients and from 20 sex- and age-matched healthy subjects. The presence of lymphocytes (CD3+ cells) within atherosclerotic plaques was determined by immunohistochemistry. Phenotypic characteristics and intracellular cytokine expression of plaque-infiltrating and circulating T lymphocytes were determined by flow cytometry. Cytokine levels in supernatants from infiltrating T cell cultures were evaluated by enzyme-linked immunosorbent assay. RESULTS: A higher number of CD3+ cells was detected in complicated than in uncomplicated plaques. Complicated plaques had higher percentages of tumor necrosis factor (TNF)-α- and interferon (IFN)-γ- positive cells than uncomplicated ones, especially in CD4+ subpopulation. In patients the percentages of TNF-α-positive cells were higher in infiltrating than in circulating lymphocyte samples. Intracellular TNF-α, IFN-γ, interleukin (IL)-4 and IL-10 expression resulted higher in circulating lymphocyte samples from patients than in those from healthy subjects. Supernatants of infiltrating T cell cultures from complicated plaques showed higher levels of TNF-α and lower levels of IL-4 than those from uncomplicated plaques. CONCLUSION: Our data provide new information on the presence of increased percentages of pro-inflammatory T lymphocytes in complicated plaques with respect to uncomplicated ones and support the concept of the key role played by activated T cells in the progression of atherosclerotic lesions.

Role of magnetic resonance imaging in the evaluation of diabetic foot with suspected osteomyelitis.

PURPOSE: This study evaluated Magnetic Resonance Imaging (MRI) in infected diabetic foot ulcers. MATERIALS AND METHODS: Sixteen diabetic patients underwent foot MRI between January 2006 and September 2007 for suspected unilateral osteomyelitis. Three of 16 patients showed radiographic changes due to Charcot neuropathic osteoarthropathy. Twelve of 16 patients also underwent MR angiography of the lower limbs for the purpose of planning surgical or endovascular treatment. The musculoskeletal and vascular MRI studies were retrospectively reviewed by three radiologists. RESULTS: The final diagnosis, based on clinical, imaging, microbiological and histological findings, was osteomyelitis in 13/16 cases. Foot MRI allowed a correct diagnosis in 15/16 patients, with 1 false positive result demonstrated by computed tomography (CT)-guided bone biopsy. MR angiography of the lower limbs was considered nondiagnostic in 5/12 patients in the infrapopliteal region owing to venous contamination. CONCLUSIONS: MRI has high sensitivity for the detection of osteomyelitis in the diabetic foot but lower specificity related to Charcot neuropathic osteoarthropathy. If diagnostic uncertainty persists, a bone biopsy is indicated. The inflammatory hyperaemia caused by the ulcer deteriorates the diagnostic quality of 40%-50% of MR angiography studies in the infrapopliteal region. In these cases, selective arteriography is appropriate, as it can be performed in the same session as angioplasty.

Stabilization of heterodimeric enzyme by multipoint covalent immobilization: Penicillin G acylase from Kluyvera citrophila.

We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)].