The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1-/- mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.     

A simple efficient synthesis of [23,24]-(13)C(2)-labeled bile salts as NMR probes of protein-ligand interactions

The synthesis of [23,24]-(13)C(2)-labeled bile salts is achieved through a steroidal side chain degradation and isotopic regeneration strategy. Three common bile acids were degraded to the corresponding C(22 )aldehyde by an oxidative decarboxylation followed by ozonolysis. The side chain was subsequently regenerated via a Horner-Emmons reaction using an ylide generated from (13)C(2)-labeled bromoacetic acid. These compounds were used as probes of protein-bile salt interactions using two- and three-dimensional NMR techniques.     

Impairment, time out of school, and time off from work after burns

Objective measurement of impairment after burns is important to patients, physicians, lawyers, and insurance companies. Even so, we could not find any references in the English literature describing how to objectively rate the physical impairment of burn survivors. The American Medical Association (AMA) has published the book Guides to Evaluation of Permanent Impairment, which is commonly used by surgeons to rate injuries. We decided to use this document to rate the impairment of burn patients. We studied patients who were treated at the University of Washington Burn Center during the years 1981, 1982, and 1983; survived the injury; were hospitalized 5 or more days or were skin grafted; and were followed until their condition was fixed (usually 12 months). This group included 325 patients. The mean age was 28.2 years and the mean total body surface area burned (TBSA) was 11.6 percent. We measured whole-man impairment (WMI) as described by the Guides to the Evaluation of Permanent Impairment. The mean whole-man impairment was 7.7 percent. In addition, we recorded time off from work and out of school after burns. The average time off from work was 12.7 weeks, and the average time out of school was 8.5 weeks. We conclude that the AMA publication can be used to rate burn patients and that the whole-man impairment of burn survivors is quite low if amputation, loss of range of motion, and nerve damage can be prevented.     

Solid-state NMR observation of cysteine and lysine Michael adducts of inactivated estradiol dehydrogenase

The inactivation of estradiol dehydrogenase by enzyme-generated 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one is accompanied by the formation of a lysine enaminone. The experiments leading to this conclusion involved degradation of the inactivated enzyme with Pronase and subsequent analysis by solution-state 13C NMR. The present paper reports solid-state 13C NMR experiments on lyophilized intact inactivated enzyme which are free from problems due to Pronase digestion. These experiments combine conventional cross-polarization and magic-angle spinning with selective irradiation of resonances arising from a 13C double label in the steroid. Magnetization transfer between neighboring 13C nuclei is used to simplify the spectra and to identify peaks due to label. The formation of cysteine and lysine Michael adducts of the enzyme is established by comparisons with chemical shifts of solid model adducts.     

Altered patterns of gene expression in Arabidopsis elicited by cauliflower mosaic virus (CaMV) infection and by a CaMV gene VI transgene

Cauliflower mosaic virus (CaMV) gene VI protein (P6) is an important determinant of symptom expression. Differential display polymerase chain reaction (PCR) was used to identify changes in gene expression in Arabidopsis elicited by a P6 transgene that causes a symptomatic phenotype. We used slot blot hybridization to measure the abundance of mRNAs complementary to 66 candidate PCR products in transgenic, CaMV-infected, and uninfected Arabidopsis plants. CaMV-infected and P6 transgenic plants showed broadly similar changes in abundance of mRNA species. In P6 transgenic plants we detected 18 PCR products that showed unambiguous changes in abundance plus another 15 that showed more limited changes (approximately twofold). CaMV-infected plants showed 17 unambiguous and 13 limited changes. Down-regulated species include those encoding a novel, phenol-like sulfotransferase, and a glycine-rich, RNA-binding protein. Up-regulated species included ones encoding an myb protein, glycine-rich and stress-inducible proteins, and a member of a previously unreported gene family. CaMV infection causes alterations in expression of many Arabidopsis genes. Transgene-mediated expression of P6 mimics virus infection in its effect on host gene expression, providing a potential mechanism for this process.