Interferon Sensitivity of Venezuelan Equine Encephalomyelitis Virus

Two strains of Venezuelan equine encephalomyelitis virus, which differ in virulence for mice, have been studied for their production of and sensitivity to chick and mouse interferon. Little interferon was produced by chick cells in response to the virulent Trinidad strain or the attenuated TC-83 strain without either aging or priming the cultures. Consistent differences in the production of chick interferon were not found between the two strains. Plaque variants of the Trinidad strain produced higher titers of mouse interferon than the TC-83 strain in both primed and control L-cell cultures. The TC-83 strain was found to be more sensitive than the Trinidad strain to the inhibitory effects of interferon. The greater sensitivity of the TC-83 strain was observed at both high and low multiplicities and for both chick and mouse interferons. These results are consistent with the hypothesis that interferon sensitivity may have a role as a determinant of virulence in some virus-host systems.     

The minimum inhibitory concentration of kitasamycin, tylosin and tiamulin for Mycoplasma gallisepticum and their protective effect on infected chicks

The minimum inhibitory concentration (m.i.c.) of kitasamycin, tylosin and tiamulin for Mycoplasma gallisepticum (Mg) were compared with 10(6), 10(4), and 10(2) CFU/ml of the organisms with the drug incorporated in mycoplasma agar. The lowest m.i.c. was obtained with tiamulin and the highest with kitasamycin and, in general, the m.i.c.'s were directly influenced by the concentration of mycoplasmas. Chick embryos at 19 days of incubation were infected with Mg and the hatched infected chicks were used for comparing the protective effect of the three drugs. They were given in the drinking water when the chicks were 2, 3 and 4 days of age. Weight gains for the infected treated birds were similar for all three drugs; they were significantly lower than for those of uninfected chicks and significantly higher than for those of untreated infected chicks. However Mg could be isolated from a high proportion of chicks in the infected treated and untreated groups at 32 days of age.     

Optimal analysis of composite cytokine responses during alloreactivity

The one-way mixed lymphocyte reaction (MLR) is a useful model of the graft-vs.-host (GvH) response that occurs following bone-marrow transplantation (BMT). Previous studies of the MLR have shown high levels of type-1 cytokine production, such as IL-1, IL-6, IFN-γ and TNF-, but low or undetectable levels of type-2 cytokines, such as IL-4 and IL-10. Here, through establishing optimal conditions for the examination of levels and kinetics of a more definitive panel of type-1/type-2 cytokines (IL-4, IL-5, IL-10 and IL-13, IFN-γ, TNF- and the soluble IL-4 receptor) we show that, contrary to previously published data, the human alloresponse is truly heterogeneous, resulting in abundant type-2 as well as type-1 cytokine secretion. The kinetics of cytokine levels in the MLR show surprising complexity, suggesting a well-defined regulation as the alloresponse develops over time. Furthermore, each MLR responder:stimulator combination tested produces a composite cytokine profile that is intrinsic to that particular pairing. These combination-specific cytokine responses are reproducible when tested on multiple occasions over time. These data reveal a potential clinical application for the cytokine MLR in selecting donors for BMT with the least inflammatory cytokine profile. Additional analysis of this system reveals that the bulk of cytokine measured is both allospecific and T-cell-derived, with comparatively low levels produced through an autologous mechanism. Interestingly, although most of the cytokine detected is produced by CD45RO+ ‘mature/activated’ T cells, CD45RA+ ‘naive’ T cells are responsible for transient early production of IL-4. This novel finding suggests that naive T cells themselves could regulate type-1/type-2 developmental fate through an autocrine IL-4 mechanism.     

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.     

Baseline levels of chromosome instability in the human lymphoblastoid cell TK6

Induced genomic instability in the human B lymphoblastoid cell line TK6 manifests itself as increases in end-to-end chromosome fusions and non-reciprocal chromosome translocations. It is not associated with elevated frequencies of specific locus mutations or other cytogenetic alterations. Previous studies on a limited number of cells and end-points suggested that induced instability in TK6 mirrors spontaneous instability in terms of the types of alterations observed. In the present study we expanded on our previous analysis to include more cells and more end-points in order to derive a more precise measure of spontaneous instability in TK6 cells. The frequency of normal growth rate thymidine kinase mutants (TK(-/-)), measured in 44 independently isolated clones, was 2.73 +/- 0.78 x 10(-6)/cell, while that for slow growth mutants was 2.39 +/- 0.52 x 10(-6)/cell. These are similar to the frequencies observed for HPRT mutants in primary human cells. There was wide variation in chromatid break frequencies, but the average break frequency, at 0.04+/-0.01 breaks/cell, was only slightly higher than that reported for primary human cells. In contrast, the dicentric frequency of 0.006/cell was more than 10-fold higher for TK6 cells than that reported for normal primary human cells. Furthermore, the dicentrics in TK6 cells are unusual in that they are the result of end-to-end chromosome fusions. TK6 cells also show much higher levels of non-reciprocal chromosome translocations than are usually observed in primary human cells. The results suggest an inherent instability in TK6 cells that differs from what is observed in primary cells in that it affects the frequency of end-to-end chromosome fusions and non-reciprocal chromosome translocations, but not TK gene mutations or other cytogenetic alterations.     

Kv11.1 channel subunit composition includes MinK and varies developmentally in mouse cardiac muscle

The Kv11.1 (also ERG1) K(+) channel underlies cardiac I(Kr), a current that contributes to repolarization in mammalian heart. In mice, I(Kr) current density decreases with development and studies suggest that changes in the structure and/or properties of the heteromultimeric I(Kr)/Kv11.1 channel are responsible. Here, using immunohistochemistry, we report that total Kv11.1 alpha subunit protein is more abundant in neonatal heart and is distributed throughout both adult and neonatal ventricles with greater abundance in epicardia. Immunoblots reveal that the alpha subunit alternative splice variant, Kv11.1a, is more abundant in adult heart while the Kv11.1b variant is more abundant in neonatal heart. Additionally, MinK channel subunit protein is shown to co-assemble with Kv11.1 protein and is more abundant in neonatal heart. In summary, Kv11.1/I(Kr) channel composition varies developmentally and the higher I(Kr) current density in neonatal heart is likely attributable to higher abundance of Kv11.1/I(Kr) channels, more specifically, the Kv11.1b splice variant.