Imaging of vulnerable atherosclerotic plaques with FDG-microPET: no FDG accumulation

BACKGROUND: Non-invasive methods of evaluating atherosclerosis in humans and experimental animals are needed. Studies indicate that FDG-PET has a potential to detect vulnerable, inflamed atherosclerotic lesions. METHODS: Nine atherosclerotic apoE-deficient mice were PET scanned. Four to determine optimal timing for imaging, and five post mortem after 1h redistribution of FDG and again after sequential removal of the interscapular brown fat and the atherosclerotic aortic arch. Uptake in various tissues in fasting (n=13) and non-fasting (n=7) apoE-deficient mice, including atherosclerotic and non-atherosclerotic aorta, was measured. Finally, accelerated atherosclerosis was induced by carotid ligation (n=12), and FDG-uptake was measured. RESULTS: FDG accumulation initially thought to correspond to the atherosclerotic aortic arch was recorded. Removal of interscapular brown fat, but not atherosclerotic aortic arch, removed the signal. The aortic arch accumulated less FDG than the non-atherosclerotic thoracic aorta both in fasting (ratio 0.5, p=0.008) and non-fasting (ratio 0.33, p=0.02) conditions. Carotid atherosclerosis likewise failed to increase FDG-uptake compared to the non-ligated artery (ratio 1.03). CONCLUSION: Spontaneously developed advanced atherosclerotic lesions in aorta were, paradoxically, associated with reduced FDG uptake, and accelerated carotid atherosclerosis also failed to increase FDG-uptake. The results seriously question the potential of FDG-PET for imagining of advanced, vulnerable atherosclerotic lesions.     

Polyuria of thyrotoxicosis: downregulation of aquaporin water channels and increased solute excretion

Thyrotoxicosis is a common disorder causing cardiovascular and renal irregularities. In this study, thyrotoxicosis was produced in rats by 14 days of daily thyroxine injection. This was associated with an increase in cardiac index, mean arterial pressure, and renal blood flow compared with euthyroid controls. Food and water intake along with urine output were significantly increased in the thyrotoxic rats compared with control animals associated with a significant increase in solute excretion. Polyuria and increased solute excretion still occurred even when food and water intake was equivalent. These renal responses were associated with significant decreases in AQP1 and AQP2 water channel expression in both the ad lib and paired intake studies in the cortex and inner medulla. The downregulation of AQP2 protein occurred in spite of equivalent plasma arginine vasopressin (AVP) in the ad lib and increased AVP in the paired feeding studies. Solute-free water reabsorption was greater in both the ad lib and paired thyrotoxic than euthyroid rats and was associated with increased Na-K-2Cl cotransporter expression. We propose that the AVP-independent downregulation of AQP2, the observed increase in renal arterial pressure, and decrease in filtration fraction contribute to polyuria the increased solute excretion in spite of enhanced ion transporters in thyrotoxicosis.     

Alemtuzumab depletes dendritic cells more effectively in blood than in skin: a pilot study in patients with chronic lymphocytic leukemia

The antibody alemtuzumab (anti-CD52) is effective in preventing acute graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (aHSCT). As well as depleting donor T cells, alemtuzumab may also work by targeting host dendritic cells (DC). To determine whether this second mechanism of action is significant, we investigated the effects of intravenous alemtuzumab by comparing skin and blood DC numbers of patients with chronic lymphocytic leukemia, before and after a 4-week course of alemtuzumab treatment. Although skin DC express CD52, the epitope is only weakly detectable and their numbers were not consistently reduced by alemtuzumab. In contrast, circulating blood DC, with stronger CD52 expression, were invariably diminished by alemtuzumab. Because DC depletion in the transplant recipient remains a promising approach for GvHD prophylaxis and therapy, more potent techniques, such as an antibody of different specificity, may be required for effective DC eradication in GvHD target organs.     

The impact of haptic learning in telemanipulator-assisted surgery

BACKGROUND: The use of a telemanipulator requires special training and surgical performance is associated with a learning curve. The aim of this study was to demonstrate the potential value of Haptic-Visual over Visual-Only passive Training in telemanipulator-assisted surgery. METHODS: Two telemanipulator consoles (da Vinci, Intuitive Surgical) were linked through an Application Programer's Interface allowing the applicant at the training console to register the position and passively follow the motions of the instructor's master telemanipulators (MTMs) at the master console (Haptic-Visual Learning group, HVL). The applicant could not actively interfere with the MTM movements. Both the trainee and the instructor shared the same 3-dimensional vision. Alternatively, subjects received only standard visual training without touching the MTMs (Visual-Only Learning group, VL). A standardized demonstration of tasks and the system was given for both groups. Participants (n=20) without previous experience with telemanipulation performed a set of various tasks in a randomized order. Study end points were time and accuracy required to perform the different task. RESULTS: The first task, with moving items to appropriate locations, showed differences in time to perform the task [mean: 4:06 min (HVL) vs. 5:16 min (VL) (P=0.2)] and accuracy differed among groups [mean number of errors 1.7 (VL) vs. 1.3 (HVL) P=0.38]. With more challenging tasks [cut out round figures (cut) and performing double dot suture lines (sti)] the number of errors was less in the HVL group [mean: 1.1 errors (cut) (P=0.05) and 1.8 errors (sti) (P=0.26)] compared with the VL group [mean: 1.8 errors (cut) and 2.3 errors (sti)]. In addition, the time to perform the tasks decreased in the HVL group with mean: 5.42 minutes (cut) (P=0.26) and 9.41 minutes (sti) (P=0.36) compared with the VL group with mean: 7.09 minutes (cut) and 11.43 minutes (sti). CONCLUSIONS: This study demonstrated the impact of haptic-visual passive learning in telemanipulator-assisted surgery which may alter the training for telemanipulator-assisted endoscopic procedures.     

Tropism of lentiviral vectors in skin tissue

The skin is an attractive tissue for gene therapy applications to treat genetic disorders and to express systemically delivered transgenes encoding therapeutic proteins. Understanding the tissue tropism of vectors is a prerequisite for the design of gene therapy trials. Using an ex vivo system of organ culture, we studied factors that determined viral tropism to the epidermal and dermal cells in human and mouse skin. We applied in these studies a lentiviral vector pseudotyped with two glycoproteins that use different cell receptors (vesicular stomatitis virus glycoprotein [VSV-G] and amphotropic murine leukemia virus envelope). The extent of infection with the amphotropic pseudotype was much higher than that of VSV-G, especially at low multiplicities of infection. In contrast, the tropism of these two pseudotypes in skin tissues was similar; at low multiplicities the infection was limited to areas near the basal layer of the epidermis, whereas at high multiplicities the infection extended to the dermal layer. To overcome physical barriers in the skin, the epidermal and dermal layers were separated and infected. Whereas the human epidermis was readily infected, we could not detect infection of stem and early progenitor cells in their niche. In contrast, mouse epidermis was completely resistant to infection. Dermal cells of both species were readily infected with the two pseudotypes. Molecular analysis indicated that infection of mouse epidermal cells was restricted after proviral DNA synthesis and before integration. In conclusion, we show that lentiviral tropism in a solid tissue is dependent on several factors, extra- and intracellular, distinct of the cellular receptors.     

Antibodies with dual reactivity to plasminogen and complementary PR3 in PR3-ANCA vasculitis

Patients with inflammatory vascular disease caused by anti-neutrophil cytoplasmic autoantibodies (ANCA) can harbor antibodies not only to the autoantigen proteinase 3 (PR3) but also to complementary PR3 (cPR3(105-201)), a recombinant protein translated from the antisense strand of PR3 cDNA. The purpose of this study was to identify potential endogenous targets of anti-cPR3(105-201) antibodies. Patients' plasmapheresis material was tested for the presence of antigens reactive with affinity-purified rabbit and chicken anti-cPR3(105-201) polyclonal antibodies. Antigen-containing fractions were tested with patients' anti-cPR3(105-201) affinity-purified IgG, and putative protein targets were sequenced by mass spectrometry. Unexpectedly, plasminogen was identified as a target of anti-cPR3(105-201). Reactivity of affinity-purified antibodies from two patients was lost when plasminogen was converted to plasmin, indicating restricted specificity. Antiplasminogen antibodies from five patients bound plasminogen at a surface-exposed loop structure within the protease domain. This loop contains an amino acid motif that is also found in a portion of recombinant cPR3(105-201); site-directed mutagenesis of this sequence decreased antibody reactivity by 30%. Functionally, antiplasminogen antibodies delayed the conversion of plasminogen to plasmin and increased the dissolution time of fibrin clots. Serologically, antiplasminogen antibody levels were higher in PR3-ANCA patients (n = 72) than healthy control subjects (n = 63), myeloperoxidase-ANCA patients (n = 34), and patients with idiopathic thrombosis (n = 57; P = 0.001). Of the patients with PR3-ANCA, nine had documented deep venous thrombosis events, five of whom were positive for antiplasminogen antibodies. In summary, capitalizing on interactions with complementary proteins, specifically complementary PR3, this study identified plasminogen as a previously undescribed autoantigen in PR3-ANCA vasculitis.     

Design of protease-resistant myelin basic protein-derived peptides by cleavage site directed amino acid substitutions

Multiple Sclerosis (MS) is considered to be a T cell-mediated autoimmune disease. An attractive strategy to prevent activation of autoaggressive T cells in MS, is the use of altered peptide ligands (APL), which bind to major histocompatibility complex class II (MHC II) molecules. To be of clinical use, APL must be capable of resisting hostile environments including the proteolytic machinery of antigen presenting cells (APC). The current design of APL relies on cost- and labour-intensive strategies. To overcome these major drawbacks, we used a deductive approach which involved modifying proteolytic cleavage sites in APL. Cleavage site-directed amino acid substitution of the autoantigen myelin basic protein (MBP) resulted in lysosomal protease-resistant, high-affinity binding peptides. In addition, these peptides mitigated T cell activation in a similar fashion as conventional APL. The strategy outlined allows the development of protease-resistant APL and provides a universal design strategy to improve peptide-based immunotherapeutics.