In vivo imaging of sterile microglial activation in rat brain after disrupting the blood-brain barrier with pulsed focused ultrasound: [18F]DPA-714 PET study.

BACKGROUND: Magnetic resonance imaging (MRI)-guided pulsed focused ultrasound combined with the infusion of microbubbles (pFUS+MB) induces transient blood-brain barrier opening (BBBO) in targeted regions. pFUS+MB, through the facilitation of neurotherapeutics' delivery, has been advocated as an adjuvant treatment for neurodegenerative diseases and malignancies. Sterile neuroinflammation has been recently described following pFUS+MB BBBO. In this study, we used PET imaging with [18F]-DPA714, a biomarker of translocator protein (TSPO), to assess for neuroinflammatory changes following single and multiple pFUS+MB sessions. METHODS: Three groups of Sprague-Dawley female rats received MRI-guided pFUS+MB (Optison™; 5-8 × 107 MB/rat) treatments to the left frontal cortex and right hippocampus. Group A rats were sonicated once. Group B rats were sonicated twice and group C rats were sonicated six times on weekly basis. Passive cavitation detection feedback (PCD) controlled the peak negative pressure during sonication. We performed T1-weighted scans immediately after sonication to assess efficiency of BBBO and T2*-weighted scans to evaluate for hypointense voxels. [18F]DPA-714 PET/CT scans were acquired after the BBB had closed, 24 h after sonication in group A and within an average of 10 days from the last sonication in groups B and C. Ratios of T1 enhancement, T2* values, and [18F]DPA-714 percent injected dose/cc (%ID/cc) values in the targeted areas to the contralateral brain were calculated. Histological assessment for microglial activation/astrocytosis was performed. RESULTS: In all groups, [18F]DPA-714 binding was increased at the sonicated compared to non-sonicated brain (%ID/cc ratios > 1). Immunohistopathology showed increased staining for microglial and astrocytic markers in the sonicated frontal cortex compared to contralateral brain and to a lesser extent in the sonicated hippocampus. Using MRI, we documented BBB disruption immediately after sonication with resolution of BBBO 24 h later. We found more T2* hypointense voxels with increasing number of sonications. In a longitudinal group of animals imaged after two and after six sonications, there was no cumulative increase of neuroinflammation on PET. CONCLUSION: Using [18F]DPA-714 PET, we documented in vivo neuroinflammatory changes in association with pFUS+MB. Our protocol (utilizing PCD feedback to minimize damage) resulted in neuroinflammation visualized 24 h post one sonication. Our findings were supported by immunohistochemistry showing microglial activation and astrocytosis. Experimental sonication parameters intended for BBB disruption should be evaluated for neuroinflammatory sequelae prior to implementation in clinical trials.

Improved Accuracy of Minimally Invasive Transpedicular Screw Placement in the Lumbar Spine With 3-Dimensional Stereotactic Image Guidance: A Comparative Meta-Analysis.

STUDY DESIGN: This study compares the accuracy rates of lumbar percutaneous pedicle screw placement (PPSP) using either 2-dimensional (2-D) fluoroscopic guidance or 3-dimensional (3-D) stereotactic navigation in the setting of minimally invasive spine surgery (MISS). This represents the largest single-operator study of its kind and first comprehensive review of 3-D stereotactic navigation in the setting of MISS. OBJECTIVE: To examine differences in accuracy of lumbar pedicle screw placement using 2-D fluoroscopic navigation and 3-D stereotaxis in the setting of MISS. SUMMARY OF BACKGROUND DATA: Surgeons increasingly rely upon advanced image guidance systems to guide minimally invasive PPSP. Three-dimensional stereotactic navigation with intraoperative computed tomography offers well-documented benefit in open surgical approaches. However, the utility of 3-D stereotaxis in the setting of MISS remains incompletely explored by few studies with limited patient numbers. MATERIALS AND METHODS: A total of 599 consecutive patients underwent minimally invasive lumbar PPSP aided by 3-D stereotactic navigation. Postoperative imaging and medical records were analyzed for patient demographics, incidence and degree of pedicle breach, and other surgical complications. A total of 2132 screw were reviewed and compared with a meta-analysis created from published data regarding the placement of 4248 fluoroscopically navigated pedicle screws in the setting of MISS. RESULTS: In the 3-D navigation group, a total of 7 pedicle breaches occurred in 6 patients, corresponding to a per-person breach rate of 1.15% (6/518) and a per-screw breach rate of 0.33% (7/2132). Meta-analysis comprised of data from 10 independent studies showed overall breach risk of 13.1% when 2-D fluoroscopic navigation was utilized in MISS. This translates to a 99% decrease in odds of breach in the 3-D navigation technique versus the traditional 2-D-guided technique, with an odds ratio of 0.01, (95% confidence interval, 0.01-0.03), P<0.001. CONCLUSIONS: Three-dimensional stereotactic navigation based upon intraoperative computed tomography imaging offers markedly improved accuracy of percutaneous lumbar pedicle screw placement when used in the setting of MISS.

Emergency ECT in an incapacitated, medically compromised patient with Huntington's disease.

Electroconvulsive therapy (ECT) is infrequently considered an "emergency" medical procedure; however, there are certain conditions in which there is considerable urgency to initiate ECT. For example, prompt administration of ECT to treat neuroleptic malignant syndrome and malignant catatonia is necessary to improve a patient's overall prognosis and potentially save the patient's life. In this case, a 57-year-old woman with Huntington's disease was admitted to our medical intensive care unit for failure to thrive due to severe psychotic symptoms. Prior to her admission, the patient had become increasingly psychotic and agitated, resulting in her refusal and/or inability to eat. Efforts to treat her severe psychiatric and behavioral symptoms with various psychopharmacological strategies were largely unsuccessful. As the patient's physical health continued to decline, with loss of approximately 35 pounds over 2 months, her family began making arrangements to transfer her to a hospice facility. The day before she was to be transferred, the psychiatry consultation-liaison service recommended ECT. Unfortunately, this recommendation was complicated because the patient was unable to provide consent. This case report describes the legal and administrative process used to ethically and legally administer ECT without consent from the patient or a court-appointed guardian in order to treat a life-threatening condition. To the best of our knowledge, this report documents the first time ECT has been granted "medical emergency" status in Texas.

The First Example of Nickel-Catalyzed Silyl-Heck Reactions: Direct Activation of Silyl Triflates Without Iodide Additives.

For the first time, nickel-catalyzed silyl-Heck reactions are reported. Using simple phosphine-supported nickel catalysts, direct activation of silyl triflates has been achieved. These results contrast earlier palladium-catalyzed systems, which require iodide additives to activate silyl-triflates. These nickel-based catalysts exhibit good functional group tolerance in the preparation of vinyl silanes, and unlike earlier systems, allows for the incorporation of trialkylsilanes larger than Me3Si.

The use of 14C-FIAU to predict bacterial thymidine kinase presence: implications for radiolabeled FIAU bacterial imaging.

UNLABELLED: Currently available infectious disease imaging techniques cannot differentiate between infection and sterile inflammation or between different types of infections. Recently, radiolabeled FIAU was found to be a substrate for the thymidine kinase (TK) enzyme of multiple pathogenic bacteria, leading to its translational use in the imaging of bacterial infections. Patients with immunodeficiencies, however, are susceptible to a different group of pathogenic bacteria when compared to immunocompetent subjects. In this study, we wanted to predict the usefulness of radiolabeled FIAU in the detection of bacterial infections commonly occurring in patients with immunodeficiencies, in vitro, prior to attempting in vivo imaging with (124)I-FIAU-PET. METHODS: We obtained representative strains of bacterial pathogens isolated from actual patients with genetic immunodeficiencies. We evaluated the bacterial susceptibility of different strains to the effect of incubation with FIAU, which would implicate the presence of the thymidine kinase (TK) enzyme. We also incubated the bacteria with (14)C-FIAU and consequently measured its rate of incorporation in the bacterial DNA using a liquid scintillation counter. RESULTS: Unlike the other bacterial strains, the growth of Pseudomonas aeruginosa was not halted by FIAU at any concentration. All the tested clinical isolates demonstrated different levels of (14)C-FIAU uptake, except for P. aeruginosa. CONCLUSION: Radiolabeled FIAU has been successful in delineating bacterial infections, both in preclinical and pilot translational studies. In patients with immunodeficiencies, Pseudomonas infections are commonly encountered and are usually difficult to differentiate from fungal infections. The use of radiolabeled FIAU for in vivo imaging of those patients, however, would not be useful, considering the apparent lack of TK enzyme in Pseudomonas. One has to keep in mind that not all pathogenic bacteria possess the TK enzyme and as such will not all retain FIAU. Our technique is simple, and can be easily used to assess whether a certain bacterial strain of interest can or cannot be visualized using radiolabeled FIAU.

Human mesenchymal stem cells inhibit endothelial proliferation and angiogenesis via cell-cell contact through modulation of the VE-Cadherin/ß-catenin signaling pathway.

Over the past 10 years, a great deal has been learned about the fundamental biology and therapeutic application of bone marrow-derived human mesenchymal stem cells (MSCs). Intravenous administration of these cells is the preferred route for therapeutic delivery of MSCs. Vascular endothelial cells (ECs) are the first cell type that MSCs encounter following IV administration. However, little is known about the biological consequences of interactions between MSCs and ECs, and if any therapeutic benefit results from this interaction. We show that MSCs exert potent stabilizing effects on ECs using an in vitro coculture system. Such effects include decreased EC proliferation and the reduction of EC vascular network formation in matrigel. Interestingly, these effects appear to require EC-MSC contact and result in enhanced colocalization of VE-Cadherin and ß-catenin at the cell membrane. Disruption of the VE-Cadherin/ß-catenin interaction abrogates the observed effects. As a functional in vivo correlate, we show that intravenously administered MSCs strongly inhibit angiogenesis in a matrigel plug assay. Taken together, these results identify a novel mechanism of action of MSCs that involves a contact-dependent EC interaction. These findings are relevant to intravenous use of MSCs and provide insight into further optimizing therapeutic strategies involving MSCs.

HIV-1 transgenic rat CD4+ T cells develop decreased CD28 responsiveness and suboptimal Lck tyrosine dephosphorylation following activation.

Impaired CD4+ T cell responses, resulting in dysregulated T-helper 1 (Th1) effector and memory responses, are a common result of HIV-1 infection. These defects are often preceded by decreased expression and function of the alpha/beta T cell receptor (TCR)-CD3 complex and of co-stimulatory molecules including CD28, resulting in altered T cell proliferation, cytokine secretion and cell survival. We have previously shown that HIV Tg rats have defective development of T cell effector function and generation of specific effector/memory T cell subsets. Here we identify abnormalities in activated HIV-1 Tg rat CD4+ T cells that include decreased pY505 dephosphorylation of Lck (required for Lck activation), decreased CD28 function, reduced expression of the anti-apoptotic molecule Bcl-xL, decreased secretion of the mitogenic lympokine interleukin-2 (IL-2) and increased activation induced apoptosis. These events likely lead to defects in antigen-specific signaling and may help explain the disruption of Th1 responses and the generation of specific effector/memory subsets in transgenic CD4+ T cells.

Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma.

Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.

Establishment of an ex vivo model of monocytes-derived macrophages differentiated from peripheral blood mononuclear cells (PBMCs) from HIV-1 transgenic rats.

It has been developed an HIV type 1 transgenic rat model (HIV-1 Tg Rat) which contains a gag-pol-deleted HIV-1 provirus regulated by the viral LTR promoter. Although it harbors a non infectious provirus, efficient viral expression occurs in different tissues and disease manifestations as well as immune-response alterations and pathologies similar to humans can be observed. Regulation of HIV-1 expression is influenced by various cellular factors and it is well known that macrophages are one of the major reservoir of HIV-1 infection and a vehicle for virus spread to other tissues. Purpose of our work was to establish an antigen presenting cells (monocyte-derived macrophages, MDM) ex vivo model from these HIV-1 transgenic rats. This model can be used to study function of HIV-infected MDM and their behavior like HIV-1 reservoir. Ultimately, these studies may be helpful in defining approaches to control HIV-1 spread.

Oocyte maturation in Xenopus laevis is blocked by the hormonal herbicide, 2,4-dichlorophenoxy acetic acid.

Oocyte maturation is dependent on a complex program of morphological, ultrastructural, and biochemical signaling events, and if disrupted could lead to decreased fertility and population decline. The in vitro sensitivity of amphibian oocytes and oocyte maturation to plant growth factor and widely used hormonal herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), was examined in this study to determine its potential impact on early development and possible contribution to the global amphibian decline. Progesterone, which acts through a membrane receptor, triggers meiotic maturation in full grown (stage VI) Xenopus oocytes, characterized by cytoskeletal reorganization, nuclear dissolution, chromosome condensation, and spindle formation. Biochemically, the Mos/MAPK/MPF signaling pathway is activated, in part dependent on translational activation of specific maternal mRNAs such as c-Mos. Light microscopy revealed unusual asymmetric morphotypes in oocytes exposed to 2,4-D alone characterized by a white spot and bulge, termed coning, in the animal pole where the germinal vesicle (nucleus) persisted intact. Treatment of oocytes with cytochalasin B, a microfilament inhibitor, blocked these morphotypes but nocodazole, a microtubule depolymerizing agent, did not. Confocal microscopy showed that 2,4-D, itself, caused substantial depolymerization of perinuclear microtubules. Importantly, 2,4-D blocked progesterone-induced maturation as measured by the lack of nuclear breakdown, confirmed by the lack of Mos expression, MPF activation, and cytoplasmic polyadenylation of cyclin B1 mRNA. However, Western blot analysis and U0126 inhibitor studies showed that 2,4-D, either alone or in the presence of progesterone, induced MAPK phosphorylation through MAPKK. These results show that 2,4-D disrupts oocyte cytoskeletal organization and blocks maturation while stimulating an independent MAPK signaling pathway.

Kaposi's sarcoma-like tumors in a human herpesvirus 8 ORF74 transgenic mouse.

The product of human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is related structurally and functionally to cellular chemokine receptors. ORF74 activates several cellular signaling pathways in the absence of added ligands, and NIH 3T3 cells expressing ORF74 are tumorigenic in nude mice. We have generated a line of transgenic (Tg) mice with ORF74 driven by the simian virus 40 early promoter. A minority (approximately 30%) of the Tg mice, including the founder, developed tumors resembling Kaposi's sarcoma (KS) lesions, which occurred most typically on the tail or legs. The tumors were highly vascularized, had a spindle cell component, expressed VEGF-C mRNA, and contained a majority of CD31(+) cells. CD31 and VEGF-C are typically expressed in KS. Tumors generally (but not always) occurred at single sites and most were relatively indolent, although several mice developed large visceral tumors. ORF74 was expressed in a minority of cells in the Tg tumors and in a few other tissues of mice with tumors; mice without tumors did not express detectable ORF74 in any tissues tested. Cell lines established from tumors expressed ORF74 in a majority of cells, expressed VEGF-C mRNA, and were tumorigenic in nude mice. The resultant tumors grew rapidly, metastasized, and continued to express ORF74. Cell lines established from these secondary tumors also expressed ORF74 and were tumorigenic. These data strongly suggest that ORF74 plays a role in the pathology of KS and confirm and extend previous findings on the tumorigenic potential of ORF74.