The Th2 cytokine genes IL4, IL5, and IL13 are clustered and expressed in a cell lineage-specific manner. We investigated the global locus-specific regulation of these genes using BAC transgenic mice containing the murine Th2 cytokine cluster carrying an IL4 promoter-luciferase reporter. IL4 promoter activity in effector CD4 T cells from these transgenic mice was strong, Th2 specific, and copy number dependent, suggesting the presence of an LCR in the locus. The production of IL4 and IL13, but not IL5, by these cells was also copy number dependent. Deletion analysis defined a 25 kb fragment in the RAD50 gene as the region containing the LCR activity. Expression of the IL4 promoter-luciferase reporter was transactivated by GATA-3 irrespective of position in the locus, suggesting the global nature of this regulation. The LCR itself, however, does not respond directly to GATA-3.

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Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the Staphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.

Capsular polysaccharides are the primary antigenic components involved in protective immunity against encapsulated bacterial pathogens. Although immunization of adolescents and adults with polysaccharide antigens has reduced pathogen disease burden, pure polysaccharide vaccines have proved ineffective at conferring protective immunity to infants and the elderly, age cohorts that are deficient in their adaptive immune responses to such antigens. However, T-cell–independent polysaccharide antigens can be converted into more potent immunogens by chemically coupling to a “carrier protein” antigen. Such “conjugate vaccines” efficiently induce antibody avidity maturation, isotype switching, and immunological memory in immunized neonates. These immune responses have been attributed to T-cell recognition of peptides derived from the coupled carrier protein. The covalent attachment of polysaccharide antigens to the carrier protein is thought to be imperative to the immunological properties of conjugate vaccines. Here we provide evidence that covalent attachment to carrier proteins is not required for conversion of T-independent antigens into T-dependent immunogens. Simple entrapment of polysaccharides or a d-amino acid polymer antigen in a cross-linked protein matrix was shown to be sufficient to produce potent immunogens that possess the key characteristics of conventional conjugate vaccines. The versatility and ease of manufacture of these antigen preparations, termed protein capsular matrix vaccines (PCMVs), will likely provide improvements in the manufacture of vaccines designed to protect against encapsulated microorganisms. This in turn could improve the availability of such vaccines to the developing world, which has shown only a limited capacity to afford the cost of conventional conjugate vaccines.