Marianne J. Skeen

The Chimpanzee as an Animal Model for Investigating Alcoholism

Young chimpanzees (Pan troglodytes) will accept ethanol in quantities sufficient to produce symptoms of withdrawal when ethanol is subsequently discontinued. Mild to severe symptoms of physical dependence, including grand mal seizures, are observed when ethanol is abruptly withdrawn after 6 to 10 weeks of chronic oral intake. In addition, the rate of disappearance of ethanol in blood increased during periods of chronic ingestion, an indication of developing metabolic tolerance. These results suggest that the young chimpanzee may be a suitable model for experimental studies of alcoholism.

Exaggerated proinflammatory and Th1 responses in the absence of gamma/delta T cells after infection with Listeria monocytogenes.

ABSTRACT While γ/δ T cells are involved in host defense and immunopathology in a variety of infectious diseases, their precise role is not yet clearly defined. In the absence of γ/δ T cells, mice die after infection with a dose of Listeriamonocytogenes that is not lethal in immunologically intact animals. Morbidity might result from insufficient levels of cytokines normally produced by γ/δ T cells or conversely from an excess of cytokines due to a lack of down-regulation of the inflammatory response in the absence of γ/δ T cells. Consistent with a regulatory role, we found that systemic levels of proinflammatory cytokines (interleukin-6 [IL-6], IL-12, and gamma interferon [IFN-γ]) were significantly higher in the absence of γ/δ T cells during the innate phase of the response. Using combinations of genetically altered and immunodepleted mice, we found evidence for γ/δ T-cell-mediated regulation of IFN-γ production by multiple cell types of both lymphoid and myeloid lineages. The antigen-specific α/β T-cell response that followed the exaggerated innate response was also increased in γ/δ T-cell-deficient mice. These findings are consistent with an emerging picture from a variety of immune response models of a critical role for γ/δ T cells in down-modulation of the immune response.

Changes in peritoneal myeloid populations and their proinflammatory cytokine expression during infection with Listeria monocytogenes are altered in the absence of γ/δ T cells

Evidence that γ/δ T cells play a broad, immunoregulatory role has been accumulating steadily. We show here that myeloid cells are disregulated after peritoneal infection with Listeria monocytogenes in mice lacking γ/δ T cells. Inflammatory populations of neutrophils and monocytes recruited to the site of infection remained longer. Intracellular cytokine analysis showed that frequencies of myeloid cells producing interleukin‐12 and tumor necrosis factor α were higher and remained elevated longer after infection in mice genetically deficient in γ/δ T cells. In vivo dye‐tracking studies indicated that the majority of inflammatory monocytes differentiated into resident tissue macrophages in situ. In vitro experiments confirmed that monocytes harvested from mice lacking γ/δ T cells were defective in their maturation process. This evidence suggests that γ/δ T cells promote differentiation in the monocyte/macrophage lineage. These cells are important for bactericidal activity, inflammatory cytokine production, clearance of inflammatory neutrophils, and ultimately, antigen presentation to T cells. Regulation of monocyte/macrophage differentiation may underlie a broad segment of the phenotypic alterations that have been reported in mice lacking γ/δ T cells.

Localization of an encephalitogenic epitope for the SJL mouse in the N-terminal region of myelin basic protein

Abstract T cells from SJL mice reactive with myelin basic protein peptide 1–38 have been reported to be encephalitogenic when adoptively transferred into naive syngeneic recipients. To determine whether the encephalitogenic epitope recognized by peptide 1–38-specific SJL T cells was different from those recognized by H-2 u -restricted MBP peptide 1–38-specific T cells, peptide 1–38-specific SJL T cell lines were developed following immunization with guinea pig MBP peptide 1–38. Following a period of in vitro selection in the presence of peptide 1–38 and syngeneic antigen-presenting cells, one ot two T cell lines transfered severe clinical disease adoptively. The second line was not encepahlitogenic. When the fine specificity for antigen of the two T cell lines was determined by the use of overlapping synthetic peptides, the encephalitogenic epitope recognized by the encephalitogenic line was loacalized to residues 17–27. This epitope is clearly distinct from that recognized by H-2 u mice. The non-encephalitogenic line was found to react only with peptide 1–38, and did not react with mouse MBP.

Protective immunity to Listeria monocytogenes elicited by immunization with heat-killed Listeria and IL-12. Potential mechanism of IL-12 adjuvanticity

The results presented here demonstrate the striking potentiating effects of IL-12 when it is combined with listerial immunogens. Although HKLM alone does not elicit strong T-cell responses, the results presented here demonstrate that the combination of HKLM and IL-12 elicited vigorous Listeria-specific Th1-type T-cell responses when administered intraperitoneally. The intensity of these responses, as well as the cytokine profiles of the Listeria-specific peritoneal T cells and macrophages, was remarkably similar to that of Listeria-infected/immune mice. These studies also revealed that typically nonimmunogenic forms of soluble listerial antigen preparations (cLLO, SLP) and LLO peptide homologs (M. A. Miller et al., manuscript in preparation) elicited intense Listeria-specific T-cell responses when administered with IL-12. In conjunction with the generation of specific T-cell responses following injection of IL-12 in combination with either killed Listeria or soluble listerial antigen preparations, macrophages from these mice expressed upregulated quantities of class II MHC and produced increased amounts of IL-12 following restimulation in vitro. Protection studies established that the Listeria-specific T-cell responses elicited by the HKLM + IL-12 mixture conferred protective immunity of mice to a lethal dose of viable L. monocytogenes. Studies designed to investigate the regulation of IL-12 production by peritoneal macrophages revealed that activated macrophages are particularly sensitive to bacterial products. However, nonviable or replication-incompetent bacteria or bacterial products injected alone were unable to influence the ability of macrophages to produce IL-12. The ability of activated macrophages to respond to HKLM was dramatically upregulated upon addition of IFN-gamma and markedly downregulated in the presence of the Th2 cytokines, IL-4 and IL-10. In light of what is known about the ability of IL-12 to induce IFN-gamma production by NK cells and gamma delta T cells, these results suggest that the exogenous addition of IL-12 may help initiate a cytokine cascade which enables the immune system to interact productively with an antigen that is typically nonimmunogenic when administered alone. These findings demonstrate that IL-12 may prove to be a powerful and broadly useful adjuvant component of particulate and soluble antigen-based vaccines directed towards many types of intracellular pathogenic microorganisms. Studies aimed at determining the generality of these findings in other infectious disease models as well as experiments designed to further elucidate the mechanism(s) of IL-12 adjuvanticity are continuing.

Induction of physical dependence of ethanol in rhesus monkeys using an oral acceptance technique

Abstract Symptoms of physical dependence such as extreme tremor and hyper-reflexia were observed in 3 female rhesus monkeys that had orally accepted doses of ethanol mixed with a totally liquid diet. These symptoms were associated with declines in blood ethanol levels following previously elevated concentrations and were manifest despite the use of a nutritionally adequate, high protein diet. In addition, ethanol elimination rates increased significantly during the ethanol administration period indicating the development of metabolic tolerance.

Role of α/β T and γ/δ T Cells in Innate and Acquired Immunity

T-lymphocyte subpopulations can be classified according to the differential expression of T-cell receptors, surface markers, adhesion molecules, cytokines, and their receptors. One distinction is in the expression of T-cell surface molecules such as CD4 and CD8. The binding of CD4 to class I1 MHC molecules or CD8 to class I MHC molecules is thought to create a requisite stabilizing “bridge” between the antigenpresenting cell and the responding T cell as well as a conduit of transducing signals. Class I MHC molecules displayed on a variety of cells are involved in antigen presentation to CD8’ T cells, whereas class I1 MHC molecules, expressed on a more restricted spectrum of cell types such as macrophages and B cells, serve as recognition molecules for CD4+ T cells. As such, the appropriate interactions of CD8’ cytotoxic T cells with infected cells and CD4’ helper cells with B cells and macrophages are facilitated. T cells of the CD4’ subset can also be subdivided into at least two major types by their patterns of cytokine production. TH1 cells secrete gamma-interferon (INF-y) and interleukin-2 (IL-2), whereas TH2 cells produce IL-4, IL-5, IL-6, and IL10. These differences in lymphokine secretion correlate with functional specialization. TH1 cells are involved in delayed-type hypersensitivity reactions and macrophage activation: whereas TH2 cells may be more important for helping B cells respond with expression of certain antibody isotypes.4 Differential regulation of these subsets plays an important role in infectious disease,’ transplantation reactions: and hypersensitivity .7

Changes in blood methanol concentrations in chimpanzees during periods of chronic ethanol ingestion.

Abstract Accumulation of blood methanol was observed in young chimpanzees ( Pan troglodytes ) during 6–14 week periods of chronic ethanol ingestion when blood ethanol levels were continually above 10–20 mg/100 ml blood. This occurred when the daily dose of ethanol was greater than that amount which could be eliminated during the 24-hr period. If these conditions were maintained, blood methanol concentrations increased for 4–5 days, then reached a plateau and remained elevated at fluctuating levels until blood ethanol concentrations decreased to less than 60-15 mg/100 ml. If ethanol concentrations continued to decline below these levels, methanoi concentrations then decreased linearly at a rate which was positively correlated with the rate of elimination of blood ethanol. Accumulation of blood methanol is probably the result of competitive inhibition of methanol oxidation by ethanol. The relationship between the observed increases in elimination rates of both ethanol and methanol and the possibility of ethanolstimulated enzymatic alterations are discussed.

Retention of functional tolerance to ethanol in rhesus monkeys (Macaca mulatta)

Tolerance to ethanol (3 g/kg 90 min prior to testing) was assessed in a group of 4 rhesus monkeys in which tolerance development had been observed using the same behavioral task one year prior to the present study. Although some decrements in performance on a two-choice discrimination reversal learning task were observed, these changes were transient and statistically insignificant. Results indicate that functional tolerance persisted throughout a one year abstinence period.

IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes

Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity. We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12. Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system. We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization. IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses. In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response. IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed.