Myeloperoxidase Activity Imaging Using <Superscript>67</Superscript>Ga Labeled Substrate

Purpose The aim of the study is to assess the feasibility of imaging specific activity of myeloperoxidase (MPO), a leukocyte enzyme with important roles in inflammation and atherosclerosis, by single photon emission computed tomography (SPECT) using a novel ⁶⁷Ga-labeled radiotracer obtained by conjugating desferrioxamine (DF) and hydroxyindolyl acetic acid in vivo. Materials and Methods A reducing substrate of MPO (I) was synthesized by reacting commercially available DF with 2-(5-hydroxy-1H-indol-3-yl) acetic acid in the presence of a coupling agent [dicyclohexyl carbodiimide (DCC)]. The chelating unit was labeled with ⁶⁷Ga, and its interaction with MPO was characterized using MALDI-TOF and UV–vis. Mice with Matrigel implants containing human MPO were used to model diseased tissues rich in MPO. Three hours after the injection of ⁶⁷Ga–I, SPECT/computed tomography (CT) imaging was performed on a high-resolution Gamma Medica X-SPECT system. Biodistribution studies were performed six hours after the injection of the radiotracer. Results The feasibility of compound I oligomerization in the presence of MPO and MPO-mediated cross-linking with proteins was initially confirmed in vitro. In vivo, a 2.7-fold increase in target-to-muscle ratio could be measured in MPO-containing Matrigel implants in mice. Biodistribution experiments demonstrated a 60% increase of radioactivity in MPO-containing vs. control (contralateral) Matrigel implants. Conclusion ⁶⁷Ga–I can be used to image MPO activity in a model system. The accumulation mechanism is based on a differential pharmacokinetics because of the size increase resulting from ⁶⁷Ga–I interaction with the target enzyme. M. Querol and J. W. Chen contributed equally to this work.     

Alzheimer''s amyloid β interaction with normal human plasma high density lipoprotein: association with apolipoprotein and lipids

We report studies of the interaction of Alzheimer's amyloid beta protein (Aβ) with normal human plasma high density lipoprotein (HDL), aiming to clarify to which lipoprotein (LP) structural constituent (apolipoprotein or lipid) soluble Aβ is primarily bound. Purified HDLs were incubated with biotinylated Aβ1–40 followed by LP repurification by size exclusion (SE) HPLC. SDS–PAGE, immunoblot and N-terminal sequence analysis of the biotin–Aβ positive protein bands revealed that Aβ is bound to many apolipoproteins of the HDL, mainly apoA-I, apoA-II, apoE and apoJ. On the other hand, reconstituted, protein-free HDL lipid particles also bind Aβ peptide and inhibit its aggregation, as intact HDL does. This was assessed by SE–HPLC, SDS–PAGE, immunoblot analysis, ultrastructural electron microscopy and Congo Red staining for β amyloid fibrils. Our data imply that Aβ binding to lipids may play an important role in maintaining the peptide in solution and thus be particularly relevant to Aβ normal and pathologic biochemistry and physiology.     

Tele-rehabilitation service delivery journey from prototype to robust in-home use

Purpose: The purpose of this study is to present a retrospective study on clients with Acquired Brain Injury (ABI) enrolled in a tele-motion-rehabilitation service program for two or more months.

Methods: Data from 82 clients (46 males; 74 with ABI), aged 22–85 years, are reported. The Kinect-based CogniMotion System (ReAbility Online, Gertner Institute, Tel Hashomer, Israel) provided services that included 30-min biweekly sessions. Participants were evaluated prior to and 2 months following the commencement of service with clinical assessments that measured movements and function of the weaker upper extremity and cognitive abilities.

Results: Clients enrolled in the service had intact or mild cognitive impairment, mild-moderate motor impairment but little use of their weak upper extremity for daily activities. They were satisfied with the service and reported high levels of system usability. Post-intervention clinical assessments were performed on about half of the participants after 2 months; significant improvements in active movements of the weak upper extremity, shoulder flexion range of motion and in the Trail Making Test were found (p?Conclusions: The service appears to be feasible for people with ABI and effective in important clinical outcomes related to improvements in upper extremity function.

• Implications for Rehabilitation

• Tele-rehabilitation provided with Microsoft Kinect 3D sensor virtual reality tracking system is feasible for people with Acquired Brain Injury.

• People with Acquired Brain Injury in the chronic stage were satisfied with the tele-rehabilitation service and perceived it as beneficial to improve their motor and cognitive abilities

• The CogniMotion System service appears to be effective in important clinical outcomes related to improvements in upper extremity function.

     

Protected Graft Copolymer (PGC) Basal Formulation of Insulin as Potentially Safer Alternative to Lantus® (Insulin-Glargine): A Streptozotocin-Induced,Diabetic Sprague Dawley Rats Study

Purpose

To develop a long-acting formulation of native human insulin with a similar pharmacodynamics (PD) profile as the insulin analogue insulin glargine (Lantus®, Sanofi-Aventis) with the expectation of retaining native human insulin’s superior safety profile as insulin glargine is able to activate the insulin-like growth factor 1 (IGF-1) receptor and is linked to a number of malignancies at a higher rate than regular human insulin.

Methods

Development of protected graft copolymer (PGC) excipients that bind native human insulin non-covalently and testing blood glucose control obtained with these formulations in streptozotocin-induced diabetic Sprague Dawley rats compared to equally dosed insulin glargine.

Results

PGC-formulations of native human insulin are able to control blood glucose to the same extent and for the same amount of time after s.c. injection as the insulin analogue insulin glargine. No biochemical changes were made to the insulin that would change receptor binding and activation with their possible negative effects on the safety of the insulin.

Conclusion

Formulation with the PGC excipient offers a viable alternative to biochemically changing insulin or other receptor binding peptides to improve PD properties.

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Figure Blood glucose development in STZ-diabetic Sprague Dawley rats after s.c. injection of 1 mg/kg regular human insulin formulated with formulations 605c, 421a, and 421b, or an equivalent dose of insulin glargine.

     

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.     

A20 deficiency in B cells enhances B-cell proliferation and results in the development of autoantibodies

A20/TNFAIP3 is an ubiquitin-editing enzyme, important for the regulation of the NF-κB pathway. Mutations in the TNFAIP3 gene have been linked to different human autoimmune disorders. In human B-cell lymphomas, the inactivation of A20 results in constitutive NF-κB activation. Recent studies demonstrate that in mice the germline inactivation of A20 leads to early lethality, due to inflammation in multiple organs of the body. In this report, we describe a new mouse strain allowing for the tissue-specific deletion of A20. We show that B-cell-specific deletion of A20 results in a dramatic reduction in marginal zone B cells. Furthermore, A20-deficient B cells display a hyperactive phenotype represented by enhanced proliferation upon activation. Finally, these mice develop higher levels of serum immunoglobulins, resulting in an excessive production of self-reactive autoantibodies.     

Microwave tomography of extremities: 2. Functional fused imaging of flow reduction and simulated compartment syndrome

Medical imaging has recently expanded into the dual- or multi-modality fusion of anatomical and functional imaging modalities. This significantly improves the diagnostic power while simultaneously increasing the cost of already expensive medical devices or investigations and decreasing their mobility. We are introducing a novel imaging concept of four-dimensional (4D) microwave tomographic (MWT) functional imaging: three dimensional (3D) in the spatial domain plus one dimensional (1D) in the time, functional dynamic domain. Instead of a fusion of images obtained by different imaging modalities, 4D MWT fuses absolute anatomical images with dynamic, differential images of the same imaging technology. The approach was successively validated in animal experiments with short-term arterial flow reduction and a simulated compartment syndrome in an initial simplified experimental setting using a dedicated MWT system. The presented fused images are not perfect as MWT is a novel imaging modality at its early stage of the development and ways of reading reconstructed MWT images need to be further studied and understood. However, the reconstructed fused images present clear evidence that microwave tomography is an emerging imaging modality with great potentials for functional imaging.     

Isatin binding proteins in rat brain: In situ imaging,quantitative characterization of specific [3H]isatin binding,and proteomic profiling

Isatin (indole‐2,3‐dione) is an endogenous indole that has a distinct and discontinuous distribution in the brain and in other mammalian tissues and body fluids. Its output is increased under conditions of stress and anxiety. Its biological targets remain poorly characterized, although [³H]isatin binding sites have been demonstrated in various brain structures. In this study, by using a real‐time beta‐imager, [³H]isatin radioligand binding analysis, and proteomic identification of proteins specifically bound to the affinity sorbent 5‐aminocaproyl‐isatin‐Sepharose, we have investigated the distribution of [³H]isatin specific binding sites in the rat brain, characterized their K_d and B_max, and identified some individual brain isatin binding proteins. The binding of [³H]isatin to rat brain sections was saturable and characterized by K_d values (of 0.2–0.3 μM) consistent with physiological concentrations. The highest B_max was found in the hypothalamus, consistent with a role in stress. In most brain regions, the homologous inhibition of [³H]isatin binding by increasing concentrations of cold isatin demonstrated complex behavior suggesting involvement of various binding proteins characterized by different affinity to isatin. Affinity chromatography of Triton X‐100 lysates of whole‐brain homogenates on 5‐aminocaproyl‐isatin‐Sepharose followed by subsequent proteomic analysis resulted in identification of 25 individual proteins, including glyceraldehyde‐3‐phosphate dehydrogenase, one of few previously reported isatin binding proteins, and a group of cytoskeleton‐related proteins. These binding sites may be related to the known antiproliferative and proapoptotic activities of isatin. © 2009 Wiley‐Liss, Inc.     

Hemorheological efficiency of drugs, targeting on intracellular phosphodiesterase activity: in vitro study

This in vitro study was designed to examine changes of red cell microrheological parameters (red cell aggregation and their suspension viscosity) after cell incubation with some drugs having phosphodiesterase (PDE) inhibitory activity (pentoxifylline - 25.0 microg/ml; drotaverine - 10.0 microg/ml; vinpocetine - 5.0 microg/ml; papaverine - 10.0 microg/ml; caffeine - 25.0 microg/ml; 3-isobutyl-1-methylxanthine [IBMX] - 10.0 microg/ml). Concentrations of used drugs for in vitro red cell microrheology study were the similar with those which it could be possible in blood of patient after intravenous therapeutic infusion. Red blood cells were separated from the blood by centrifugation at 1400 g for 15 min and washed 3 times with phosphate buffered saline (PBS). The washed RBCs were then resuspended in PBS at a hematocrit of approximately 40%. In each of the research sessions these RBC suspensions were divided into two aliquots and exposed to: one of the drug at 37 degrees C for 15 min; remaining aliquot (red cell suspension with PBS) was kept at 37 degrees C for 15 min and served as the control. It was found that all of used drugs decreased red cell aggregation and their suspension viscosity significantly. Since IBMX and vinpocetine are the specific inhibitor PDE activity it might be suppose that cellular PDE is molecular target in RBCs for this class of drugs. The obtained data reveals evidence that drugs, acting as PDE inhibitors, might be considered as microrheologically positive remedies.