Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis.

BackgroundEndovascular technological advances have revolutionized the field of neurovascular surgery and have become the mainstay of treatment for many cerebrovascular pathologies. Digital subtraction angiography (DSA) is the ’gold standard' for visualization of the vasculature and deployment of endovascular devices. Nonetheless, with recent technological advances in optics, angioscopy has emerged as a potentially important adjunct to DSA. Angioscopy can offer direct visualization of the intracranial vasculature, and direct observation and inspection of device deployment. However, previous iterations of this technology have not been sufficiently miniaturized or practical for modern neurointerventional practice.ObjectiveTo describe the evolution, development, and design of a microangioscope that offers both high-quality direct visualization and the miniaturization necessary to navigate in the small intracranial vessels and provide examples of its potential applications in the diagnosis and treatment of cerebrovascular pathologies using an in vivo porcine model.MethodsIn this proof-of-concept study we introduce a novel microangioscope, designed from coherent fiber bundle technology. The microangioscope is smaller than any previously described angioscope, at 1.7 F, while maintaining high-resolution images. A porcine model is used to demonstrate the resolution of the images in vivo.ResultsVideo recordings of the microangioscope show the versatility of the camera mounted on different microcatheters and its ability to navigate external carotid artery branches. The microangioscope is also shown to be able to resolve the subtle differences between red and white thrombi in a porcine model.ConclusionA new microangioscope, based on miniaturized fiber optic technology, offers a potentially revolutionary way to visualize the intracranial vascular space.

BACKGROUND Delta-24-RGD, an oncolytic adenovirus, shows promise against glioblastoma. To enhance virus delivery, we recently demonstrated that human bone marrow-derived mesenchymal stem cells loaded with Delta-24-RGD (hMSC-D24) can eradicate glioblastomas in mouse models. There are no studies examining the safety of endovascular selective intra-arterial (ESIA) infusions of MSC-D24 in large animals simulating human clinical situations. OBJECTIVE To perform canine preclinical studies testing the feasibility and safety of delivering increasing doses of hMSCs-D24 via ESIA infusions. METHODS ESIA infusions of hMSC-D24 were performed in the cerebral circulation of 10 normal canines in the target vessels (internal carotid artery [ICA]/P1) via transfemoral approach using commercially available microcatheters. Increasing concentrations of hMSC-D24 or particles (as a positive control) were injected into 1 hemisphere; saline (negative control) was infused contralaterally. Toxicity (particularly embolic stroke) was assessed on postinfusion angiography, diffusion-weighted magnetic resonance imaging, clinical exam, and necropsy. RESULTS ESIA injections were performed in the ICA (n = 7) or P1 (n = 3). In 2 animals injected with particles (positive control), strokes were detected by all assays. Of 6 canines injected with hMSC-D24 through the anterior circulation, escalating dose from 2 × 106 cells/20 mL to 1 × 108 cells/10 mL resulted in no strokes. Two animals had ischemic and hemorrhagic strokes after posterior cerebral artery catheterization. A survival experiment of 2 subjects resulted in no complications detected for 24-h before euthanization. CONCLUSION This novel study simulating ESIA infusion demonstrates that MSCs-D24 can be infused safely at least up to doses of 1 × 108 cells/10 mL (107 cells/ml) in the canine anterior circulation using commercially available microcatheters. These findings support a clinical trial of ESIA infusion of hMSCs-D24.

How to cite this article: Bikhet MH, Hansen-Estruch C, Javed M, et al. Profound thrombocytopenia associated with administration of multiple anti-inflammatory agents in baboons.

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