Gene and antisense delivery in alcoholism research.

This article represents the proceedings of a symposium at the 2001 annual meeting of the Research Society on Alcoholism in Montreal, Canada. Drs. Yedy Israel and Fulton Crews were organizers and co-chairpersons. The presentations were (1) Introduction to the symposium, by Yedy Israel; (2) Gene delivery to the brain, by Fulton T. Crews; (3) Gene therapy in alcoholic liver injury, by Ronald Thurman; and (4) Antisense oligonucleotides and antisense-gene delivery, by Yedy Israel.

B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation.

Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freund's adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freund's adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.

Functional consequences of dysregulated B7-1 (CD80) and B7-2 (CD86) expression in B or T lymphocytes of transgenic mice.

T cell activation and tolerance are regulated by interactions between CD28 or CTLA-4 on T cells and B7 costimulatory molecules on APCs. We have generated transgenic mouse strains that constitutively express B7-1 (CD80) at high levels on B cells or T cells or express B7-2 (CD86) on T lymphocytes to examine the consequences of dysregulated B7 expression on T cell responses. The transgene-derived B7 molecules are functional, because B7-1 transgenic B cells are more efficient APCs than are wild-type B cells, and the activation of B7 transgenic T cells is less dependent on exogenous costimulation than that of wild-type T cells. In vivo, constitutive expression of B7 molecules leads to the elimination of immature B cells. The expression of B7 molecules on thymocytes results in the down-regulation of CD28 expression. However, B7 transgenic mice have normal numbers of mature lymphocytes and mount normal T cell responses following immunization with protein Ag. Neither anergy induction nor superantigen-mediated deletion of T cells is altered by the dysregulated expression of B7-1 or B7-2 on B or T lymphocytes in these transgenic strains. Therefore, functionally significant levels of B7 expressed constitutively on mature lymphocytes are not, by themselves, sufficient to abrogate T cell tolerance or induce autoimmune disease.

Coexpression of B7-1 and antigen blocks tolerance induction to antigen presented by resting B cells.

To investigate a role for B cells as tolerogenic APCs in peripheral lymphoid organs, we have developed a system in which B cells from mice transgenic for the membrane-bound form of human mu-chain are transferred into nontransgenic recipients. Mice injected with B cells expressing human mu-chain became profoundly tolerant to human mu-chain, as shown by greatly reduced Ab responses following challenge with human mu-chain in adjuvant. Adoptive transfer experiments showed that the recipient's Th cell response to human mu-chain was impaired. When the human mu transgenic spleen cells were activated with LPS before transfer, they no longer induced tolerance. Spleen cells from double-transgenic mice expressing both human mu-chain and the costimulatory molecule B7-1 (CD80) also failed to induce tolerance to human mu-chain. However, neither human mu transgenic LPS blasts nor double-transgenic B cells induced an Ab response or primed for a secondary Ab response to Ag in adjuvant. Therefore, we find that expression of B7-1 together with Ag can interfere with tolerance induction without inducing Ab formation or priming for a secondary Ab response.

Efficient expression of protein coding genes from the murine U1 small nuclear RNA promoters.

Few promoters are active at high levels in all cells. Of these, the majority encode structural RNAs transcribed by RNA polymerases I or III and are not accessible for the expression of proteins. An exception are the small nuclear RNAs (snRNAs) transcribed by RNA polymerase II. Although snRNA biosynthesis is unique and thought not to be compatible with synthesis of functional mRNA, we have tested these promoters for their ability to express functional mRNAs. We have used the murine U1a and U1b snRNA gene promoters to express the Escherichia coli lacZ gene and the human alpha-globin gene from either episomal or integrated templates by transfection, or infection into a variety of mammalian cell types. Equivalent expression of beta-galactosidase was obtained from < 250 nucleotides of 5'-flanking sequence containing the complete promoter of either U1 snRNA gene or from the 750-nt cytomegalovirus promoter and enhancer regions. The mRNA was accurately initiated at the U1 start site, efficiently spliced and polyadenylylated, and localized to polyribosomes. Recombinant adenovirus containing the U1b-lacZ chimeric gene transduced and expressed beta-galactosidase efficiently in human 293 cells and airway epithelial cells in culture. Viral vectors containing U1 snRNA promoters may be an attractive alternative to vectors containing viral promoters for persistent high-level expression of therapeutic genes or proteins.

A negative regulatory function of B7 revealed in B7-1 transgenic mice.

To analyze the functions of T cell costimulators in vivo, we have constructed a transgenic mouse strain that constitutively expresses murine B7-1 on mature B cells. Antibody responses to T-dependent hapten-protein conjugates and serum immunoglobulin levels are markedly depressed in B7-1 transgenic mice. This immune deficiency is not due to an intrinsic B cell defect, as antibody responses to T-independent hapten conjugates are normal. Furthermore, treatment with anti-B7-1 restores the capacity of transgenic mice to respond to hapten-protein conjugates, demonstrating that the deficient antibody responses are directly attributable to the expression of B7-1. These results suggest that the temporally regulated expression of costimulators such as B7-1 may contribute to either initiation or down-regulation (feedback inhibition) of T-dependent immune responses in vivo, and that the inhibitory function is dominant in transgenic mice that constitutively express high levels of this costimulator.

Mutational analysis of the herpes simplex virus type 1 glycoprotein E promoter.

A set of linker-scanning mutations was constructed in the herpes simplex virus type 1 (HSV-1) glycoprotein E (gE) promoter to define cis-acting regulatory elements common to late viral promoters. The gE promoter is a late viral promoter that has some activity in the absence of viral DNA replication and is representative of the gamma 1 class of HSV-1 promoters. Each promoter mutation was inserted upstream of the Escherichia coli lacZ gene in a recombinant virus, and the relative activities of beta-galactosidase expressed from individual recombinant viruses were compared. This analysis identified two sequence elements, a TATA element and an element at the start of transcription, that corresponded to similarly positioned elements previously identified in the gC and gH late (gamma 2) HSV-1 promoters. Mutation of the initiation element reduced expression from this promoter during normal lytic infection, but had no appreciable effect on expression in the absence of viral DNA replication, suggesting a specific role in late gene expression. Analysis of expression from hybrid gE/gC promoters revealed that the TATA and the initiation elements of these two promoters were interchangeable and that expression from the gE promoter in the absence of viral DNA replication was due to regulatory elements upstream from TATA element.

The 9-kDa hydrophobic protein encoded at the 3' end of the porcine transmissible gastroenteritis coronavirus genome is membrane-associated.

The open reading frame potentially encoding a 78 amino acid, 9101 Da hydrophobic protein (HP) and, mapping at the 3' end of the porcine transmissible gastroenteritis coronavirus (TGEV) genome, was shown to be expressed during virus replication. The cloned HP gene was placed in a plasmid under control of the T7 RNA polymerase promoter and in vitro translation of transcripts generated in vitro yielded a 9.1-kDa protein that was immunoprecipitable with porcine hyperimmune anti-TGEV serum. Antiserum raised in rabbits against a 31 amino acid synthetic polypeptide that represented the central hydrophilic region of HP specifically immunoprecipitated HP from TGEV-infected cells. HP was further shown to become associated with microsomal membranes during synthesis in vitro and was found to be closely associated with the endoplasmic reticulum and cell surface membranes in infected cells. The intracellular location of HP suggests that it may play a role in the membrane association of replication complexes or in virion assembly.

Immune modulation within the brain: recruitment of inflammatory cells and increased major histocompatibility antigen expression following intracerebral injection of interferon-gamma.

Intracerebral injections of interferon-gamma (IFN-gamma) have multiple immunological effects on rat brain, affecting all anatomic compartments. Lymphocytes and other inflammatory cells are recruited to the injection site: CD4+ T-cells into the perivascular space, OX42+ monocytes/macrophages into brain parenchyma. IFN-gamma also recruits OX8+ cells into brain parenchyma. These OX8+ cells are not stained by 'pan' T-cell antibodies, however, suggesting that they may be natural killer cells. IFN-gamma also causes increased major histocompatibility complex expression on brain cells: class I antigen on local endothelial and ependymal cells, and class II antigen on microglial, ependymal, and perivascular cells throughout both hemispheres of the brain.

An insertion vector for the analysis of gene expression during herpes simplex virus infection.

A herpes simplex virus type 1 (HSV-1) insertion vector, pGal8, was designed for analysis of herpesvirus promoters during virus infection. This vector contains a multiple cloning site (MCS) positioned at the 5' end of the lacZ gene for the insertion of promoter sequences. The MCS and lacZ are flanked by sequences from the HSV-1 thymidine kinase encoding gene (tk) to direct homologous recombination into the tk locus of the viral genome. The utility of this vector is demonstrated by construction and comparison of recombinant viruses that express lacZ from the promoters of the genes encoding glycoprotein C, glycoprotein H and glycoprotein E.

Successful treatment of massive carbamazepine overdose.

Overdose of carbamazepine (CBZ) can be fatal. We report the case of a patient with near-lethal toxicity due to delayed absorption of drug. A 36-year-old woman was admitted with coma, hypotension, and unusual movements. Carbamazepine (CBZ) level several hours later was 36 mg/L. Gastric lavage revealed no pill fragments, and activated charcoal was administered. CBZ level initially fell, reaching 28 mg/L 36 h after admission. Blood level then rose sharply, reaching 54 mg/L 64 h after admission. The pattern of rise suggested renewed absorption of drug. Vigorous cathartics were given, and further doses of charcoal were administered. Three hours after onset of diarrhea, roving eye movements occurred. Two hours later she grimaced to pain. Eight hours after the onset of diarrhea, she was awake. In CBZ overdose, activated charcoal therapy coupled with aggressive intestinal purging helps prevent continued absorption of drug, late exacerbation of symptoms, and potentially fatal outcome.