A Novel Modular Headmount Design for non-invasive Scalp EEG Recordings in Awake Animal Models.

We have designed and developed a novel, noninvasive modular headmount to be used for awake animal scalp electroencephalography (EEG). The design is based on a developing rat that will accommodate rapid head growth. Desired characteristics include non-invasiveness, adjustable quantity and positioning, light weight, and tolerability by the animal. Axial Dependent Modular Electrode Mount (ADMEM), as designed here, addresses the aforementioned constraints by using light-weight and adjustable materials. The initial prototype of ADMEM has been tested in vivo with rat pups, using the open field test to assess for stress and anxiety at two post-installation time-points: one day after ADMEM installation (acute time-point) and four days after ADMEM installation (sub-acute time-point). There was no significant difference in normal developmental weight gain between Control and ADMEM rat groups. Although no significant difference was found in the level of anxiety between groups at the acute time-point, the ADMEM group spent significantly less time in the center of the open field test, suggesting higher anxiety. The test also showed no difference in the measured traveled distances between Control and ADMEM groups on either time-points.

3D imaging of sex-sorted bovine spermatozoon locomotion, head spin and flagellum beating.

With the advent of sperm sex sorting methods and computer-aided sperm analysis platforms, comparative 2D motility studies showed that there is no significant difference in the swimming speeds of X-sorted and Y-sorted sperm cells, clarifying earlier misconceptions. However, other differences in their swimming dynamics might have been undetectable as conventional optical microscopes are limited in revealing the complete 3D motion of free-swimming sperm cells, due to poor depth resolution and the trade-off between field-of-view and spatial resolution. Using a dual-view on-chip holographic microscope, we acquired the full 3D locomotion of 235X-sorted and 289 Y-sorted bovine sperms, precisely revealing their 3D translational head motion and the angular velocity of their head spin as well as the 3D flagellar motion. Our results confirmed that various motility parameters remain similar between X- and Y-sorted sperm populations; however, we found out that there is a statistically significant difference in Y-sorted bovine sperms' preference for helix-shaped 3D swimming trajectories, also exhibiting an increased linearity compared to X-sorted sperms. Further research on e.g., the differences in the kinematic response of X-sorted and Y-sorted sperm cells to the surrounding chemicals and ions might shed more light on the origins of these results.

Recombinant factor VIIa in trauma patients without coagulation disorders.

Recombinant activated factor VIIa (rFVIIa) has many clinical applications for patients with congenital bleeding disorders and in a variety of clinical settings. Additional studies in the future are ongoing and should provide the clinical anesthesiologist an additional option during certain bleeding states. Specific recommendations as to timing of administration and frequent monitoring of ionized calcium status are suggested at this time. Optimization of fibrinogen levels, platelet levels, pH, and body temperature will enhance efficacy of rFVIIa.

Plasmin immunization preferentially induces potentially prothrombotic IgG anticardiolipin antibodies in MRL/MpJ mice.

OBJECTIVE: To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome. METHODS: BALB/cJ and MRL/MpJ mice were immunized with Freund's complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin-immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro. RESULTS: Plasmin-immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while plasmin-immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG antiplasmin or IgG aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to beta(2)GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III, and 2 of 10 inhibited inactivation of FXa by antithrombin III. CONCLUSION: Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti-beta(2)GPI in MRL/MpJ mice, but leads to production of only IgG antiplasmin in BALB/cJ mice. IgG mAb generated from plasmin-immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive potentially prothrombotic aCL in genetically susceptible individuals.

Novel autoantibodies against the activated coagulation factor IX (FIXa) in the antiphospholipid syndrome that interpose the FIXa regulation by antithrombin.

We previously reported that some human antiphospholipid Abs (aPL) in patients with the antiphospholipid syndrome (APS) bind to the homologous enzymatic domains of thrombin and the activated coagulation factor X (FXa). Moreover, some of the reactive Abs are prothrombotic and interfere with inactivation of thrombin and FXa by antithrombin (AT). Considering the enzymatic domain of activated coagulation factor IX (FIXa) is homologous to those of thrombin and FXa, we hypothesized that some aPLs in APS bind to FIXa and hinder AT inactivation of FIXa. To test this hypothesis, we searched for IgG anti-FIXa Abs in APS patients. Once the concerned Abs were found, we studied the effects of the Ab on FIXa inactivation by AT. We found that 10 of 12 patient-derived monoclonal IgG aPLs bound to FIXa and that IgG anti-FIXa Abs in APS patients were significantly higher than those in normal controls (p < 0.0001). Using the mean + 3 SD of 30 normal controls as the cutoff, the IgG anti-FIXa Abs were present in 11 of 38 (28.9%) APS patients. Importantly, 4 of 10 FIXa-reactive monoclonal aPLs (including the B2 mAb generated against beta(2)-glycoprotein I significantly hindered AT inactivation of FIXa. More importantly, IgG from two positive plasma samples were found to interfere with AT inactivation of FIXa. In conclusion, IgG anti-FIXa Ab occurred in approximately 30% of APS patients and could interfere with AT inactivation of FIXa. Because FIXa is an upstream procoagulant factor, impaired AT regulation of FIXa might contribute more toward thrombosis than the dysregulation of the downstream FXa and thrombin.

Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi.

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.

CD4(+) (and not CD25+) T cells are the predominant interleukin-10-producing cells in the circulation of filaria-infected patients.

BACKGROUND: Interleukin (IL)-10 plays an important role in down-regulating the immune response to filarial parasites. The goal of this study was to characterize the predominant cellular source of IL-10 in human filarial infections. METHODS: Multicolor flow cytometry was used to determine the frequencies of IL-10 production from various lymphocyte populations in the circulation of 23 patients with filarial infections and 8 uninfected control subjects. RESULTS: The frequencies of cells spontaneously producing IL-10 was significantly greater in filaria-infected patients than in uninfected control subjects (geometric mean, 93 vs. 18 IL-10-producing cells/100,000 peripheral blood mononuclear cells; P = .03). Most IL-10-producing cells in filaria-infected patients were T cells, with CD4(+) and CD8(+) cells accounting for 48% and 27%, respectively, of all IL-10-producing cells; CD19(+) B cells, CD14(+) monocytes, and CD56(+) NK cells accounted for 10%, 8%, and 7%, respectively. Surprisingly, only 12% of the IL-10-producing CD3(+)CD4(+) cells were CD25(+). Seventy-seven percent of IL-10-producing CD4(+) T cells stained negatively for both IL-4 and interferon (IFN)-gamma, 22% were positive for IL-4, and <1% were positive for IFN-gamma. CONCLUSIONS: These experiments demonstrate that the most frequent producers of IL-10 in human filarial infections are CD4(+) T cells, many of which are skewed toward a type 2 phenotype and most of which are not CD25(+).