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This research determined the in vitro, intraperitoneal injection and dietary immunostimulatory effects on the grouper Epinephelus malabaricus of chitosan and its derivatives with different molecular weight, chitosan, polyglucosamine and N-acetyl-chitooligosaccharides. Respiratory burst activity of head-kidney phagocytes isolated from the grouper incubated in vitro with the chitosans at a range of concentrations was studied. Respiratory burst activity generally decreased with increasing dosage of chitosan products. N-acetyl-chitooligosaccharides were significantly more potent in enhancing respirtatory burst activity than the other two chitosans. Respiratory burst activity of head-kidney phagocytes of the grouper injected with three kinds of chitosans at 4 dosages was assayed. N-acetyl-chitooligosaccharides caused significantly higher respiratory burst activity than the other two chitosans. N-acetyl-chitooligosaccharides at the dosage of 10 µg/g was found to enhance the highest respiratory burst activity among treatments. In the time series assay with intraperitoneal injection by N-acetyl-chitooligosaccharides at a dosage of 10 µg/g, it was found that enhancement of NBT reduction occurred early in the time course of the study and is similar to the time series response of the glucan treatment. When the groupers (120g) were fed with diets containing 5 concentrations of N-acetyl-chitooligosaccharides including 0, 0.5, 1, 1.5, and 2 g/100g and stocked in indoor closed recirculation systems for 7 weeks, weight gain of the fish was not significantly affected by the dietary treatments. The immune status measured by respiratory burst activity, alternative complement pathway, agglutination titer, lysozyme activity and superoxide dismutase activity was not significantly affected by N-acetyl-chitooligosaccharides supplement. But feeding the grouper with N-acetyl-chitooligosaccharides at 1 g/100g diet seems to lower the immunity of the fish, although the effects were statistically insignificant.

Assessing ecological risk to wildlife exposed to anthropogenic contamination in soil has traditionally been problematic. Attempts to standardize an approach to evaluate risk for various community types in North America have been challenging, given the variation in terrestrial communities and the values in which policy makers are bound to protect. This has resulted in vague, yet flexible guidance from the U.S. Environmental Protection Agency and other interested parties (e.g., the U.S. Army Corps of Engineers, and the Tri-Service Ecological Risk Assessment Working Group). Interpretation of these and other guidance has been variable, often resulting in conflicting opinions on how best to address the question of ecological risk to receptors that are exposed to xenobiotics in a soil matrix. This work reports the results of research designed to address the question of ecological risk to terrestrial vertebrates. Objective, ecologically-relevant criteria were used in the selection and development of models in this research. Several lines of logic were considered: 1) substance sensitivity, 2) ecological sensitivity (i.e., the species importance to the system; e.g., keystone species); and, 3) probability and extent of exposure. A primary soil contaminant at many U.S. Army installations is 2,4,6-trinitrotoluene (TNT). This was a result of the mass manufacturing, storing, and assembly of weapons from the early 1900's until the 1950s. The Army has reported soil concentrations of TNT ranging from 0.12 to 38,600 ug/g (Walsh and Jenkins 1992) and 0.08 to 64,000 ug/g (Hovatter et al. 1997). The chemical-physical properties of TNT result in a relatively unique compound, not easily amenable to current modeling techniques to estimate exposure to terrestrial wildlife. Moreover, there are few data describing the effects of exposure to TNT in other than mammals, fish, and specific invertebrates. In this research, the pathways of exposure and selected potential toxic effects from TNT exposure were investigated in a terrestrial salamander: Ambystoma tigrinum (tiger salamanders). A. tigrinum was chosen since they are exclusively carnivorous, relatively long-lived, have a thin integument, and are large enough to investigate individual effects. These investigations were designed to mimic natural conditions as closely as possible, though maintain a degree of homogeneity in a laboratory environment. All studies exposed salamanders to soil and food (earthworms) in identical preparations. As such, these exposures were considered complete, eliminating assumptions made regarding daily food consumption, systemic dermal dose, etc. The first study examined the relative contribution of dermal or oral exposures to the whole-body burdens of TNT and primary metabolites. A poly-chlorinated biphenyl (PCB) mixture (Aroclor7 1260) was used with TNT to simultaneously to assist in the evaluation of each pathway, since the fate and transport of PCBs are well characterized. Tiger salamanders were exposed 28 days in situ. The dermal route of exposure contributed the most to the final burdens of TNT in salamanders, with the primary reduction products, 2-amino-4,6-dinitrotoluene and 4-amino, 2,6-dinitrotoulene reaching higher concentrations than of parent compound. Other TNT metabolites were found in insignificant quantities. The concentrations of PCBs were higher in the oral treatment, as expected. These results were corroborated in a subsequent study using Ambystoma maculatum (spotted salamanders). The second series of investigations evaluated the potential toxic effects from TNT exposure. Two treatments consisting of TNT and a control were used to evaluate these effects to A. tigrinum. The salamanders were exposed in situ for 14 days to TNT in soil and food (earthworms of which were exposed to TNT in the soil in similar preparations). Non-specific immune effects were evaluated through the characterization of splenic phagocytes in their ability to: 1) phagocytize foreign particles, and 2) digest (through oxygen radicals) phagocytized material. This was conducted using fluorescent microspheres and a fluorescent chemical probe specific to hydrogen peroxide, measured per each cell using flow cytometry. Other data collected included histological examination (e.g., liver, kidney, and other miscellaneous organs), blood differentials, weight changes over time, organ/ body weight comparisons, and an analysis of organ-specific metabolism. No significant effects were noted in salamanders exposed to these conditions. Coordinated with the preceding study included a search for biomarkers of exposure and an investigation of the metabolites of TNT in situ. Biotransformation products of TNT were found including primary (e.g., 2-amino-4,6-dinitrotoluene) and secondary (e.g., 2,4-diamino-6-nitrotoluene) in relative concentrations in skin, liver, and kidney. Biomarkers of exposure included an analysis of cytochrome p450, b5, and the glutathione antioxidant enzymes in liver, kidney, skin, lung, and serum, respectively. Traces of parent compound were found in the skin and liver only. Levels of 2,4-diamino-6-nitrotoluene were found only in the liver and kidney, suggesting that TNT is reduced primarily in or on the skin. Levels of p450 were higher in TNT exposed salamanders than controls. Glutathione and related enzyme levels are reported. This work suggests that salamanders have levels of detoxification enzymes capable of the biotransformation of anthropogenic substances in soil rivaling that of mammals. Another investigation evaluated these same immunological parameters in white-footed mice (Peromyscus leucopus). This species was chosen based on the relative importance of small mammals to the community structure in many North American ecosystems. Mice were exposed to TNT in the feed at 0.264, 0.066, 0.033, and 0.017%, where actual daily dose estimates for males were 604, 275, 109, and 65; and for females was 544, 282, 143, and 70 mg/kg/d. An investigation to evaluate the specificity of commercially-available monoclonal antibodies specific to cell surface markers for thymocytes and splenocytes in inbred mice was unsuccessful. These results suggest the recognition epitopes of monoclonal antibodies prepared against Old-World mice are not conserved into Peromyscus, a New-World species. However, high dose males and females had larger spleens consistent with the hemolytic effects previously reported for mammals exposed to TNT. Further, males exposed at all levels had reduced phagocytic activity of splenocytes, and reduced hydrogen peroxide production associated with the two highest doses relative to controls. Females showed no response relative to treatment. This research has shown the feasibility for these types of investigations, and provides toxicity information valuable for modeling estimates of ecological risk. Further, the in situ exposures have provided media concentrations that are or are not toxic for species of concern. This type of information reduces the uncertainty associated with ingestion modeling estimates, dermal exposure estimates, and other factors not traditionally considered in toxicity studies. / Ph. D.

During homeostatic expansion, peripheral T cells proliferate in response to lymphopenia, in the absence of cognate antigen or costimulatory signals. Host CD8 reconstitution following hematopoietic cell transplantation (HCT) involves the de novo-generation of T cells in addition to the homeostatic expansion of mature donor T cells present in the graft and donor lymphocyte infusion as well as host T cells that survive conditioning. Although it is well appreciated that CD8 homeostatic expansion contributes to host CD8 reconstitution following HCT, the factors governing the extent of the contribution by donor CD8 homeostatic expansion to host reconstitution have not been precisely and systematically examined. The ex vivo generation of memory CD8 T cells specific for an epitope of the immunodominant minor histocompatibility antigen H60 was demonstrated. By adapting a previously described culture system designed to generate large numbers of transgenic memory CD8 T cells, memory CD8 cells (TM) specific for a physiological antigen were elicited from a heterogeneous population of CD8 T cells. The ex vivo-generated antigen-specific memory CD8 cells were then expanded under conditions of lymphopenia in ablatively conditioned syngeneic transplant recipients and found to persist greater than 2 months post-transplant. These findings support the notion that the transplantation of small numbers ex vivo-generated memory CD8 cells, specific for a physiologically relevant antigen, can help restore host immune function following HCT. Transplant conditions were found to modulate the contribution of homeostatically expanded donor CD8 TM to the host CD8 compartment. Varying the conditioning intensity, timing of infusion, and infusion dose affected the kinetics of expansion as well as the homeostatic set-point. In my HCT model, delayed infusions of 3 weeks and transplantation of small numbers of donor CD8 T cells resulted in significant contributions to host CD8 T cell compartment. These findings could be applied clinically to enhance the effectiveness of clinical immunotherapy in restoring host immune function post-transplant. Moreover, transplantation of varying doses of donor CD8 TM demonstrated there was maximal donor contribution to host CD8 reconstitution. Finally, the homeostatic expansion, persistence, and function of transgenic memory and naïve CD8 populations were investigated following transplantation into ablatively conditioned syngeneic recipients. Both donor CD8 populations underwent 2 weeks of expansion post-transplant. The naïve CD8 population reproducibly achieved higher homeostatic numbers than the memory CD8 population. By one month post-HCT, the transplanted naïve CD8 populations also exhibited a memory CD8 phenotype. Moreover, the CD8 TN→TM population mediated an effective response to a primary challenge, comparable to the antigen-experienced memory CD8 population. Thus, regardless of the state of differentiation of the donor CD8 population at the time of transplant, donor CD8 homeostatic expansion contributes a memory CD8 cells population, able to mediating effective immune responses, to host reconstitution.

The aim of this thesis is to improve the therapeutic effect of the immunotherapy based on the synergy of TLR agonists with phagocytosis stimulating ligands. Furthermore, this thesis is focused on the information transfer to the specific immunity, as well as it pursues the study of the specific immunity relevance during cancer immunotherapy.

Immunologische Toleranz ist eine elementare Eigenschaft des Immunsystems, die primär durch die klonale Deletion autoreaktiver T-Zellen im Thymus gewährleistet wird. Neben diesem als zentrale Toleranz bezeichneten Mechanismus, verfügt ein Organismus gleichzeitig über periphere Toleranzmechanismen wie Ignoranz, Anergie und regulatorische T-Zellen. Trotz dieser Kontrollmechanismen können in bestimmten Situationen autoreaktive CD4+ und CD8+ T-Zellen aktiviert werden und meistens zu örtlich und zeitlich begrenzten Autoimmunreaktionen führen. Ursache hierfür kann die hormonelle Regulation oder das gewebespezifische Vorkommen eines Selbsttantigens sein. Am Beispiel von HSP60-kreuzreaktiven CD8+ T-Zellen konnte gezeigt werden, dass der Transfer dieser T-Zellen in Tiere zu einer Entzündung des Dünndarms aber nicht des Dickdarms führt, obwohl das Selbstantigen im letzteren wesentlich stärker exprimiert wird. Die Gewebespezifität der Autoimmunpathologie konnte durch die in den Organen unterschiedliche, proteasomale Antigenprozessierung, erklärt werden. Proteinbiochemische und immunologische Analysen ergaben, dass sich die 20S Proteasomen verschiedener Organe strukturell und funktionell deutlich unterscheiden und somit jedes Gewebe ein individuelles Repertoire von MHC-Klasse I restringierten Peptiden präsentiert. Damit wurde ein weiterer Mechanismus entdeckt, durch den Reaktivität von protektiven und pathologischen CD8+ T-Zellen kontrolliert wird. / Immunological tolerance which is primarily mediated by the clonal deletion of autoreactive T cells in the thymus is a key feature of the immune system. Besides this central tolerance, several mechanisms act also in the periphery including ignorance, anergy and regulatory T cells. Despite all these checkpoints, autoreactive CD4+ and CD8+ T cells may still be activated causing local and time restricted autoimmune-reactions. This may refer primarily to self-antigens which are hormonally regulated or tissue-specifically expressed. Adoptive transfer of crossreactive, hsp60-specific CD8+ T cells into mice induced an local inflammation of the small intestine but not the colon despite elevated expression of hsp60 in the latter organ. The pathology could be explained by the finding that the proteasomal antigen processing varies between different organs. Biochemical and immunological analyses revealed that 20S proteasomes of different organs vary in their structural and functional properties indicating that every tissue displays an individual and distinct repertoire of MHC class I peptides. This represents a new mechanism by which the activity of protective and pathological CD8+ T cell responses may be controlled.