Real-time imaging of Rab3a and Rab5a reveals differential roles in presynaptic function

We investigated the roles of two Rab-family proteins, Rab3a and Rab5a, in hippocampal synaptic transmission using real-time fluorescence imaging. During synaptic activity, Rab3a dissociated from synaptic vesicles and dispersed into neighbouring axonal regions. Dispersion required calcium-dependent exocytosis and was complete before the entire vesicle pool turned over. In contrast, even prolonged synaptic activity produced limited dispersion of Rab5a. A GTPase-deficient mutant, Rab3a (Q81L), dispersed more slowly than wild-type Rab3a, and decreased the rate of exocytosis and the size of the recycling pool of vesicles. While overexpression of Rab3a did not affect vesicle recycling, overexpression of Rab5a reduced the recycling pool size by 50%. We propose that while Rab3a preferentially associates with recycling synaptic vesicles and modulates their trafficking, Rab5a is largely excluded from recycling vesicles.     

Delayed development of humoral immunity in chickens following in ovo treatment with mibolerone

In order to determine if mibolerone causes rapid development of the humoral immune system as previously hypothesized, 12-day-old chicken embryos were injected with 0.1, 1, or 10 micrograms mibolerone. Mibolerone caused bursal atrophy in a dose-dependent manner with severe atrophy at the 10 micrograms dose, substantial atrophy at the 1 microgram dose, and very little or no atrophy at the 0.1 microgram dose. Mibolerone-treated chicks were subsequently challenged with sheep red blood cells (SRBC) and Brucella abortus at 1, 7, 14, or 21 days of age. When serum samples were analysed for agglutinins to SRBC and B. abortus, the results indicated that treated chickens had lower agglutinin titers to both antigens at younger ages. These responses improved with age in groups treated with 0.1 microgram and 1 microgram and were comparable to controls at 5 weeks of age except for low primary and secondary (IgG specific) titers to B. abortus in birds treated with 1 microgram. Chickens treated with 10 micrograms mibolerone still had considerably lower primary responses to SRBC and lower primary as well as secondary responses to B. abortus at 4 and 5 weeks of age. These results indicate that mibolerone causes delayed rather than rapid development of the humoral immune system. In control as well as in treated chickens, agglutinin responses to SRBC appeared earlier than responses to B. abortus.     

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4⁺ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155^−/−) mice. Infection was controlled significantly quicker in the miR155^−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155^−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ⁺CD8⁺ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155^−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155^−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155^−/− T cells was significantly enhanced in L. major−infected miR155^−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.¹ CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.^2,3 It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,⁴ whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.⁵ Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.⁴ Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.^6,7 In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.⁴miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,^8,9 and up-regulation of miR155 in CD4⁺ T cells promotes preferential Th1 differentiation and IFN-γ production¹⁰ by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11, 12, 13, 14 Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.¹⁵ miR155 has also been shown to play a role in regulating effector Th2 response.16, 17, 18 Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155^−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response.     

Imaging biomarkers of inflammation <Emphasis Type="Italic">in situ</Emphasis> with fun ctionalized quantum dots in the dextran sodium sulfate (DSS) model of mouse colitis

Objective and design: Myeloperoxidase (MPO) and proinflammatory cytokines play an important role in the development of inflammation. These markers are generally measured using tedious ELISA procedures. In this study, a novel technique utilizing antibody conjugated quantum dot nanoparticles was developed to detect Myeloperoxidase, Interleukin-1α (IL-1α) and Tumor Necrosis Factor-α (TNF-α) in vivo in the dextran sodium sulfate (DSS) model of experimental colitis. Materials and methods: Colitis was induced in animals (n = 8 animals/group) by feeding 4% DSS solution ad libitum for seven to eight days. Quantum Dots (QDs) exhibiting fluorescence at various wavelengths were conjugated to MPO, IL-1α and TNF-α polyclonal antibodies and tested in vivo at various stages of colitis. Tissue sections obtained were imaged with confocal microscope. The image intensity obtained from the tissue specimen was correlated with clinical activity measured as Disease Activity Index (DAI). Results: Myeloperoxidase, IL-1α and TNF-α were visualized with quantum dots on various days of disease. The intensity of quantum dots increased with the increase in inflammation. The increase in intensity showed an excellent correlation with the DAI based on the clinical parameters. Conclusion: The study demonstrated that multiple biomarkers can be detected simultaneously and their quantitative expression correlated well with clinical disease severity. This novel technology should facilitate design of a novel optical platform for imaging various biomarkers of inflammation, early detection of acute and chronic disease markers and inflammation-mediated cancer markers. This detection may also facilitate determination of therapeutic success. Received 14 March 2007; returned for revision 8 May 2007; accepted by M. Parnham 27 June 2007     

Optimal field splitting for large intensity-modulated fields

The multileaf travel range limitations on some linear accelerators require the splitting of a large intensity-modulated field into two or more adjacent abutting intensity-modulated subfields. The abutting subfields are then delivered as separate treatment fields. This workaround not only increases the treatment delivery time but it also increases the total monitor units (MU) delivered to the patient for a given prescribed dose. It is imperative that the cumulative intensity map of the subfields is exactly the same as the intensity map of the large field generated by the dose optimization algorithm, while satisfying hardware constraints of the delivery system. In this work, we describe field splitting algorithms that split a large intensity-modulated field into two or more intensity-modulated subfields with and without feathering, with optimal MU efficiency while satisfying the hardware constraints. Compared to a field splitting technique (without feathering) used in a commercial planning system, our field splitting algorithm (without feathering) shows a decrease in total MU of up to 26% on clinical cases and up to 63% on synthetic cases.     

Influence of lamella features of UHMWPE on its physical and uniaxial tensile properties. I. Effect of sterilization method in uncrosslinked and unaged materials

The changes in the tensile toughness (U), degree of crystallinity (%C), melting temperature (Tm), lamella thickness, and lamella alignment of uncrosslinked and unaged GUR 1150HP ultra-high-molecular-weight polyethylene specimens, following different sterilization treatments (none, gamma-irradiation in air, gamma-irradiation in a nitrogen-rich atmosphere, ethylene oxide gas, and gas plasma) were determined. For each of the properties - U, %C, and Tm - the only significant difference in the mean value was found between the set of specimens gamma-irradiated in air, on the one hand, and each of the other sets, on the other hand. It was found that lamella thickness showed little change between the groups of specimens but there was a significant difference in the lamella orientation between the set of specimens that had been gamma-irradiated in air, on the one hand, and each of the other sets, on the other. It thus appears that the changes seen in the physical and mechanical properties determined may be a reflection of the change in the polymer's lamella alignment.