The role of IFN in respiratory syncytial virus pathogenesis

Formalin-inactivated respiratory syncytial virus (RSV) vaccine preparations have been shown to cause enhanced disease in naive hosts following natural infection. In this study we demonstrate a similar pattern of enhanced disease severity following primary RSV infection of IFN-nonresponsive STAT1(-/-) mice. STAT1(-/-) mice showed markedly increased illness compared with wild-type BALB/c animals following RSV inoculation despite similar lung virus titers and rates of virus clearance. Histologically, STAT1(-/-) animals had eosinophilic and neutrophilic pulmonary infiltrates not present in wild-type or IFN-gamma(-/-)-infected mice. In cytokine analyses of infected lung tissue, IFN-gamma was induced in both STAT1(-/-) and wild-type mice, with preferential IL-4, IL-5, and IL-13 induction only in the STAT1(-/-) animals. Eotaxin was detected in the lungs of both wild-type and STAT1(-/-) mice following infection, with a 1.7-fold increase over wild-type in the STAT1(-/-) mice. Using a peptide epitope newly identified in the RSV fusion protein, we were able to demonstrate that wild-type memory CD4(+) T cells stimulated by this peptide produce primarily IFN-gamma, while STAT1(-/-)CD4(+) cells produce primarily IL-13. These findings suggest that STAT1 activation by both type I (alphabeta) and type II (gamma) IFNs plays an important role in establishing a protective, Th1 Ag-specific immune response to RSV infection.     

An efficient in situ hybridization protocol for multiple tissue sections and probes on miniaturized slides

To facilitate detection of gene activity in tissue sections we combined common protocols of in situ hybridization on tissue sections (TSISH) with the technique of whole-mount in situ hybridization (WMISH). Miniature glass slides for mounting tissue sections were cut from regular microscope slides and handled for in situ hybridization in laboratory-made 2-ml containers (baskets) similar to those originally used for WMISH on Drosophila embryos. A salient point of the method is the use of airtight reaction vessels placed in a dry thermostat for critical hybridization steps as this facilitates reproducible and stringent hybridization conditions which are difficult to achieve on tissue sections otherwise. The practicability of the method is illustrated on consecutive serial frozen sections of murine neonatal cerebellum hybridized for math1 and neuroD, two developmentally regulated genes with distinct expression patterns. For both genes excellent spatial resolution and a highly dynamic range of signal intensity was obtained. The approach enables simple processing of multiple probes, allows the efficient and economic use of small tissue samples and is amenable to automation.     

Multispecific CD4+ T cell response to a single 12-mer epitope of the immunodominant heat-shock protein 60 of Yersinia enterocolitica in Yersinia-triggered reactive arthritis: overlap with the B27-restricted CD8 epitope, functional properties, and epitope presentation by multiple DR alleles

Yersinia heat-shock protein 60 (Ye-hsp60) has recently been found to be a dominant CD4 and CD8 T cell Ag in Yersinia-triggered reactive arthritis. The nature of this response with respect to the epitopes recognized and functional characteristics of the T cells is largely unknown. CD4+ T cell clones specific for Ye-hsp60 were raised from synovial fluid mononuclear cells from a patient with Yersinia-triggered reactive arthritis. and their specificity was determined using three recombinant Ye-hsp60 fragments, overlapping 18-mer synthetic peptides as well as truncated peptides. Functional characteristics were assessed by cytokine secretion analysis in culture supernatants after specific antigenic stimulation. Amino acid positions relevant for T cell activation were detected by single alanine substitutions within the epitopes. Fragment II comprising amino acid sequence 182-371 was recognized by the majority of clones. All these clones were specific for peptide 319-342. Th1 clones and IL-10-secreting clones occurred in parallel, sometimes with the same fine specificity. The 12-mer core epitope 322-333 is a degenerate MHC binder and is presented to some T cell clones in a "promiscuous" manner. This epitope is almost identical with a B27-restricted CTL epitope of Ye-hsp60. Cross-reactivity of Ye-hsp60-specific T cell clones with self-hsp60 was not observed. In conclusion, an interesting Ye-hsp60 T cell epitope has been identified and characterized. It remains to be determined whether this epitope is also relevant in other reactive arthritis patients.     

Substrate specificities and 13-cis-retinoic acid inhibition of human, mouse and bovine cis-retinol dehydrogenases

Recent studies of the human, mouse and bovine genes for 11-cis-retinol dehydrogenase (11cRDH) and human and mouse 9-cis-retinol dehydrogenase (9cRDH) suggest that they are homologs of the same enzyme. This conclusion is inconsistent with earlier literature indicating that 11cRDH is expressed solely in the eye and does not utilize 9-cis-retinol as a substrate. We have compared directly the kinetic properties of recombinant human and mouse 9cRDH with those of bovine 11cRDH for 9-cis- and 11-cis-retinol and investigated the inhibitory properties of 13-cis-retinoic acid on each of these enzymes. Human and mouse 9cRDH and bovine 11cRDH have very similar kinetic properties towards 9-cis- and 11-cis-retinol oxidation and they respond identically to 13-cis-retinoic acid inhibition. Our biochemical data are consistent with the conclusion that 9cRDH and 11cRDH are the same enzyme.     

Aerosols transmit prions to immunocompetent and immunodeficient mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrP(C), efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrP(C) selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrP(Sc) and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.     

Phylogenetic analysis of family 6 glycoside hydrolases

Multiple sequence alignment separates members of glycoside hydrolase Family 6 into eight subfamilies: one of mainly actinobacterial endoglucanases (EGs), one of ascomycotal EGs, one of chytridiomycotal EGs and cellobiohydrolases (CBHs), one of actinobacterial and proteobacterial CBHs, one of chytridiomycotal CBHs, two of ascomycotal CBHs, and one of basidiomycotal CBHs. Each also has some proteins of unknown function. Multiple sequence alignment also extends to all of Family 6 the observation that lengths of loops that form the active-site tunnel in CBHs vary among subfamilies, and along with loop conformations, determine enzyme function.