Canine microglial cells: stereotypy in immunophenotype and specificity in function?

Microglial cells represent the endogenous immune system of the central nervous system (CNS). Upon pathological insults they reveal their immunological potential aimed at regaining homeostasis. These reactions have long been believed to follow a uniform and unspecific pattern which is irrespective to the underlying disease entity. Evidence is growing that this view seriously underrates microglial competence as the defenders of the CNS. In the present study, microglial cells of 47 dogs were examined ex vivo by means of flow cytometry. Ex vivo examination included immunophenotypic characterization using eight different surface markers and functional studies such as phagocytosis assay and the reactive oxygen species (ROS) generation test. The dogs were classified according to their histopathological diagnoses in disease categories (controls, canine distemper virus (CDV) induced demyelination, other diseases of the CNS) and results of microglial reaction profiles were compared. Immunophenotypic characterization generally revealed relative high conformity in the microglial disease response among the different groups, however the functional response was shown to be more specific. Dogs with intracranial inflammation and dogs with demyelination showed an enhanced phagocytosis, whereas a significant up-regulation of ROS generation was found in dogs with demyelination due to CDV infection. This strongly suggests a specific response of microglia to infection with CDV in the settings of our study and underlines the pivotal role of microglial ROS generation in the pathogenesis of demyelinating diseases, such as canine distemper.

[Guidance of interventions in subintimal recanalization and fenestration of dissection membranes using a novel dual-lumen intravascular ultrasound catheter]

PURPOSE: To evaluate the feasibility and effectiveness of IVUS-guided puncture for gaining controlled target lumen reentry in subintimal recanalization of chronic iliac/femoral artery occlusions and in fenestration of aortic dissections. MATERIALS AND METHODS: Between 5/2004 and 12/2005 12 consecutive patients (7 male, 5 female; mean age 64.6 +/- 12.0 years) with chronic critical limb ischemia and ischemic complications of aortic dissection were treated using the Pioneer catheter. This 6.2-F dual-lumen catheter combines a 20-MHz IVUS transducer with a pre-shaped extendable, hollow 24-gauge nitinol needle. This coaxial needle allows real-time IVUS-guided puncture of the target lumen and after successful reentry a 0.014" guidewire may be advanced through the needle into the target lumen. 7 patients were treated for aortic dissection and 5 patients (with failed previous attempts at subintimal recanalization) for chronic arterial occlusion. Patients with aortic dissection (5 type A dissections, 2 type B dissections) had developed renal ischemia (n = 2), renal and mesenteric ischemia (n = 2), or low extremity ischemia (n = 3). Patients with chronic arterial occlusions (2 common iliac artery occlusions, 3 superficial femoral artery occlusions) experienced ischemic rest pain (n = 4), and a non-healing foot ulcer (n = 1). RESULTS: The technical success rate using the Pioneer catheter was 100%. The recanalization/fenestration time was 37 +/- 12 min. Procedure-related complications did not occur. In 10 cases a significant improvement of clinical symptoms was evident. One patient with aortic dissection and ischemic paraplegia required subsequent surgical intervention. One patient had persistent ischemic rest pain despite successful recanalization of a superficial femoral artery occlusion. CONCLUSION: The Pioneer catheter is a reliable device which may be helpful for achieving target lumen reentry in subintimal recanalization of chronic occlusions and in fenestration of aortic dissections.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Differential release of plasminogen activator inhibitors (PAIs) during dual perfusion of human placenta: implications in preeclampsia

Plasminogen activator inhibitors (PAIs) play critical roles in regulating cellular invasion and fibrinolysis. An increase in the ratio of PAI-1/PAI-2 in placenta and maternal serum is suggested to result in excessive intervillous fibrin deposition and placental infarction in pregnancies complicated by preeclampsia (PE) and intrauterine growth restriction (IUGR). In the current study we used dual (maternal and fetal) perfusion of human term placentas to examine the release of PAIs to the intervillous space. ELISA revealed a significant time-dependent increase in total PAI-1 levels in maternal perfusate (MP) between 1 and 7h of perfusion. Conversely, PAI-2 levels decreased resulting in a 3-fold increase in the PAI-1/PAI-2 ratio in MP. Levels of PAI-1, but not PAI-2, in placental tissue extracts increased during perfusion. In perfusions carried out with xanthine and xanthine oxidase (X + XO), compounds used to generate reactive oxygen species (ROS), no time-dependent increase in total PAI-1 levels was observed. In addition, X + XO treatment promoted a 3-fold reduction in active PAI-1 levels in MP, indicating that ROS decrease PAI-1 release to MP. The finding of a time-dependent change in patterns of PAI expression and response to ROS indicates the utility of dual perfusion as a model to dissect mechanism(s) promoting aberrant fibrinolysis in pregnancies complicated by PE and IUGR.

Peroxide bond-dependent antiplasmodial specificity of artemisinin and OZ277 (RBx11160)

Using nonperoxidic analogs of artemisinin and OZ277 (RBx11160), the strong in vitro antiplasmodial activities of the latter two compounds were shown to be peroxide bond dependent. In contrast, the weak activities of artemisinin and OZ277 against six other protozoan parasites were peroxide bond independent. These data support the iron-dependent artemisinin alkylation hypothesis.

Single-exposure dual-energy subtraction chest radiography: detection of pulmonary nodules and masses in clinical practice

The purpose of this retrospective study was to evaluate the impact of energy subtraction (ES) chest radiography on the detection of pulmonary nodules and masses in daily routine. Seventy-seven patients and 25 healthy subjects were examined with a single exposure digital radiography system. Five blinded readers evaluated first the non-subtracted PA and lateral chest radiographs alone and then together with the subtracted PA soft tissue images. The size, location and number of lung nodules or masses were registered with the confidence level. CT was used as standard of reference. For the 200 total lesions, a sensitivity of 33.5-52.5% was found at non-subtracted and a sensitivity of 43.5-58.5% at energy-subtracted radiography, corresponding to a significant improvement in four of five readers (p < 0.05). However, in three of five readers the rate of false positives was higher with ES. With ES, sensitivity, but not the area under the alternative free-response receiver operating characteristics (AFROC) curve, showed a good correlation with reader experience (R = 0.90, p = 0.026). In four of five readers, the diagnostic confidence improved with ES (p = 0.0036). We conclude that single-exposure digital ES chest radiography improves detection of most pulmonary nodules and masses, but identification of nodules <1 cm and false-positive findings remain a problem.

Ultra-high-resolution dual-source CT for forensic dental visualization-discrimination of ceramic and composite fillings

Dental identification is the most valuable method to identify human remains in single cases with major postmortem alterations as well as in mass casualties because of its practicability and demanding reliability. Computed tomography (CT) has been investigated as a supportive tool for forensic identification and has proven to be valuable. It can also scan the dentition of a deceased within minutes. In the present study, we investigated currently used restorative materials using ultra-high-resolution dual-source CT and the extended CT scale for the purpose of a color-encoded, in scale, and artifact-free visualization in 3D volume rendering. In 122 human molars, 220 cavities with 2-, 3-, 4- and 5-mm diameter were prepared. With presently used filling materials (different composites, temporary filling materials, ceramic, and liner), these cavities were restored in six teeth for each material and cavity size (exception amalgam n = 1). The teeth were CT scanned and images reconstructed using an extended CT scale. Filling materials were analyzed in terms of resulting Hounsfield units (HU) and filling size representation within the images. Varying restorative materials showed distinctively differing radiopacities allowing for CT-data-based discrimination. Particularly, ceramic and composite fillings could be differentiated. The HU values were used to generate an updated volume-rendering preset for postmortem extended CT scale data of the dentition to easily visualize the position of restorations, the shape (in scale), and the material used which is color encoded in 3D. The results provide the scientific background for the application of 3D volume rendering to visualize the human dentition for forensic identification purposes.

Dual induction of PKR with E2F-1 and IFN-alpha to enhance gene therapy against hepatocellular carcinoma

Overexpression of the transcription factor E2F-1 induces apoptosis in tumor cells. This apoptotic effect is partly mediated through the induction of the double-stranded RNA-activated protein kinase (PKR). Here, we investigate if agents that upregulate PKR could enhance the apoptotic effect of E2F-1 overexpression in liver tumors. In human hepatocellular carcinoma (HCC) cells (Hep3B, HepG2, Huh7), adenovirus-mediated overexpression of E2F-1 (AdCMV-E2F) transcriptionally increased PKR mRNA. The subsequent increase of total and phosphorylated PKR protein was followed by induction of apoptosis. When AdCMV-E2F was combined with the PKR modifier interferon alpha (IFNalpha), PKR was additionally upregulated and both PKR activation and apoptosis were increased. Subcutaneous xenograft tumors were selectively targeted using an adenoviral vector expressing E2F-1 under the control of the human telomerase reverse transcriptase (hTERT) promoter (AdhTERT-E2F). Weekly systemic administration of AdhTERT-E2F inhibited tumor growth. The tumor suppressive effect of AdhTERT-E2F therapy was further enhanced in combination with IFNalpha.Our results demonstrate that PKR activating agents enhance the anti-tumor effect of E2F-1 overexpression in HCC in-vitro and in-vivo. Hence, modulation of PKR is a potential strategy to increase the efficacy of PKR-dependent anti-tumor therapies.

Study of and design procedure for dual circularly polarized waveguide slot arrays

For the past sixty years, waveguide slot radiator arrays have played a critical role in microwave radar and communication systems. They feature a well-characterized antenna element capable of direct integration into a low-loss feed structure with highly developed and inexpensive manufacturing processes. Waveguide slot radiators comprise some of the highest performance—in terms of side-lobe-level, efficiency, etc. — antenna arrays ever constructed. A wealth of information is available in the open literature regarding design procedures for linearly polarized waveguide slots. By contrast, despite their presence in some of the earliest published reports, little has been presented to date on array designs for circularly polarized (CP) waveguide slots. Moreover, that which has been presented features a classic traveling wave, efficiency-reducing beam tilt. This work proposes a unique CP waveguide slot architecture which mitigates these problems and a thorough design procedure employing widely available, modern computational tools. The proposed array topology features simultaneous dual-CP operation with grating-lobe-free, broadside radiation, high aperture efficiency, and good return loss. A traditional X-Slot CP element is employed with the inclusion of a slow wave structure passive phase shifter to ensure broadside radiation without the need for performance-limiting dielectric loading. It is anticipated this technology will be advantageous for upcoming polarimetric radar and Ka-band SatCom systems. The presented design methodology represents a philosophical shift away from traditional waveguide slot radiator design practices. Rather than providing design curves and/or analytical expressions for equivalent circuit models, simple first-order design rules – generated via parametric studies — are presented with the understanding that device optimization and design will be carried out computationally. A unit-cell, S-parameter based approach provides a sufficient reduction of complexity to permit efficient, accurate device design with attention to realistic, application-specific mechanical tolerances. A transparent, start-to-finish example of the design procedure for a linear sub-array at X-Band is presented. Both unit cell and array performance is calculated via finite element method simulations. Results are confirmed via good agreement with finite difference, time domain calculations. Array performance exhibiting grating-lobe-free, broadside-scanned, dual-CP radiation with better than 20 dB return loss and over 75% aperture efficiency is presented.

Algorithms for autonomous tandem operation of a dual M113 system

In mid-July 2003, the U.S. Army Tank-Automotive & Armaments Command (TACOM) performed a series of experiments at Keweenaw Research Center (KRC), with a remote operated mine roller system. This system, named Panther Lite, consists of two M113 Armored Personnel Carriers (APC’s) connected by a Tandem Vehicle Linkage Assembly (TVLA). The system has three sets of mine rollers, two of which are connected to the front of the lead vehicle with one set trailing from the trail vehicle. Currently, the system requires two joystick controllers. One regulates the braking of the tracks, throttle, and transmission of the lead vehicle and the other controls the braking and throttle of the rear vehicle. One operator controls both joysticks, attempting to maneuver the lead vehicle along a desired path. At the same time, this operator makes compensation maneuvers to reduce lateral loads in the TVLA and to guide the rear mine rollers along the desired path. The purpose of this project is to create algorithms that would allow the slave (trail) vehicle to operate using inputs that maneuver the control (lead) vehicle. The project will be completed by first reconstructing the experimental data. Kinematic models will be generated and simulations created. The models will then be correlated with the reconstructions of the experimental data. The successful completion of this project will be a first step to eliminating the need for the second joystick.

Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity

Human maltase-glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 A) and in complex with acarbose (to 1.9 A). Structural analysis of the NtMGAM-acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose.