Judy M. Teale - US grants

This proposal is designed to delineate and characterize the regulatory mechanism involved in the control of murine immunoglobulin class expression employing B cell cloning assays that facilitate the class switch and restricted or cloned helper T cell populations. Initially, we will continue to examine the role of the B cell in determining isotype expression by analyzing the extent to which B cells exhibit class commitment. This will be done by reducing problems associated with B cell heterogeneity through the separation of primed and unprimed B cells into defined B cell subsets based on s-Ig (e.g. s-IgA, s-IgE), Lyb surface antigens, and Fc receptors. In addition, we will examine the potential for isotype expression of these defined B cell subsets depending upon the nature of the stimulus by using T-dependent and T-independent forms of the antigen. We will then explore the functional relationship between these defined B cell subsets and helper T cells that are involved in the regulation of isotype expression. The parasite-infected mouse model provides a biological system whereby isotypic regulation by T cells may be maximal since these mice produce abnormally high levels of IgE. We will characterize the helper T cell population from such mice with particular emphasis on helper T cells that may be functionally restricted for the IgE isotype. The most powerful approach for determining the role of T cells in the class switch is the use of clonally derived helper T cells. We intend to clone helper T cells to a variety of carriers including parasite antigens and use these lines in B cell cloning assays with defined B cell subsets. Moreover, employing enrichment procedures we will try to generate isotype-specific T cells from both antigen-primed and parasite-infected mice. We will then define the nature of the target B cells as well as possible recognition elements involved when these functionally-restricted T cells are operative. The proposed studies should substantially contribute to our knowledge of the control mechanisms involved in immunoglobulin class production.

The mechanism of B cell differentiation and the development of the immune repertoire will be studied under defined conditions using an in vitro fetal organ culture system and long term lymphocyte cultures. B cell development and diversification in culture will be assessed by a number of criteria including 1) selection of VH genes by in situ hybridization using radiolabeled VH gene family probes, 2) frequency of hapten-responsive B cells to a number of haptens using the splenic focus assay, 3) acquisition of B cell surface markers, 4) development of predominant clonotypes by idiotypic analysis and VH gene selection, 4) degree of clonal expansion by measuring increased frequencies of already existing clonotypes, and 5) degree of diversification by examining the rate of appearance of clonotypes normally acquired late in ontogeny. Since these experiments are performed in the absence of circulation and cell migration, newly developing B cells that are uninfluenced by their environment are examined providing a unique opportunity to analyze genetic vs environmental factors affecting repertoire expression. A number of factors will be tested for their effect on B cell differentation and diversification including growth stimuli, anti-idiotypes, anti-light chain, and anti-pre B cell marker reagents. Moreover, using the criteria above, the metabolic requirements of diversification will be assessed by adding various metabolic inhibitors to the cultures. In addition, the importance of age and compartmentalization on B cell development will be further analyzed by comparing the B cell precursor pools from fetal liver, fetal spleen, and adult bone marrow. Finally, the selection of VH genes in the expressed vs available repertoire will be compared and the importance of chromosomal location of VH gene families in relation to their use in the functional repertoire evaluated.

This proposal is designed to examine the potential bioeffects of ultrasound on B cell development using both in vivo and in vitro methodologies. Specifically, the effects of ultrasound on B cell development, antibody class expression, and the diversification of B cell specificities in the developing fetus will be monitored. Pregnant Balb/c mice will be sham-treated or exposed to ultrasound radiation and sacrificed at several times during the gestation period. Fetal livers and spleens will then be dissected and analyzed directly or cultured first in an in vitro fetal organ culture system. Both uncultured and cultured fetal lymphoid cells will then be tested for the proportion of surface immunoglobulin cells (B cells), cytoplasmic Mu positive cells (pre B cells), and pre B cell marker positive cells by immunocytochemical staining. In addition, cultured and uncultured fetal lymphoid cells obtained from normal vs ultrasound exposed animals will be antigenically stimulated in the splenic focus assay and the resulting B cell clones analyzed for isotype production. Finally, cultured and uncultured fetal cells of various ages of gestation will be tested for the frequency and kinetics of appearance of B cells responsive to several different haptens. B cells specific for these haptens have been previously shown to arise in a predictable temporal order. Moreover, the appearance of individual clonotypes and the process of VH selection will be explored. If differences as a result of ultrasound are observed, B cell development will also be examined in neonatal mice. Finally, it will be determined if there are critical periods of sensitivity to ultrasound exposure. These studies should detect potential effects of ultrasound on B cell development, an important analysis given the widespread clinical use of ultrasound radiation.

A hallmark of autoimmune disease is an increase in the incidence of self-reactive B lymphocytes with the subsequent production of autoantibodies. Although it seems clear that regulatory defects are intimately associated with the onset, if not the continuance, of autoimmunity, there is little information on whether there are fundamental differences in the B cell precursor pool. One of the primary goals of this proposal is to determine if, in fact, there is a skewing of the available B cell specificity repertoire toward autoantigens or some discernable alteration in the composition of the B cell repertoire prior to the onset of autoimmunity. This will be accomplished by analyzing the immune repertoire of autoimmune vs normal strains of mice at various ages for the frequency of B cells 1) reactive with foreign and autoantigens, 2) expressing predominant idiotypes, and 3) using and expressing certain VH and VL gene families. This will be accomplished by using B cell cloning assays idiotype inhibition assays, and in situ hybridization with radiolabeled VH and VL gene probes. In addition, innate differences in the B cell precursor pool will be explored using long term bone marrow cultures. Several autoimmune mouse models and their normal counterparts will be used including NZB, MRL/lpr/lpr, and BXSB. These studies should provide significant new insights into the mechanisms of the autoimmune disease process and the involvement of the B lymphocyte lineage.

The long term goal of this proposal is to understand how antibody classes are regulated in the context of parasite immunity . Mice infected with the metacestode, Mesocestoides corti develop a hypergammaglobulinemia that is restricted to IgM and IgG1. From previous experiments, it became clear that much of the immune response is directed against molecules released from the organism. At least two of these molecules are stress proteins. As the molecules released from the organism can be used to induce an isotype restricted response in vitro, the specific aims of the proposal are designed to define associated mechanisms. First sufficient quantities of M. corti stress proteins/released molecules will be isolated using biochemical techniques and generating expression libraries. Using purified proteins, the IgG1 inducing factor will be identified. Preliminary results indicate an active release of M. corti stress proteins. Because of the known importance of stress proteins in immunity to pathogens and the potential impact of secreted molecules, the mechanism of release will be analyzed using a number of drug inhibitors. Another aim will take advantage of the large number of B cells that are activated to produce IgG1. These cells will be analyzed simultaneously for cell size and morphology , cell surface activation markers, and expression of gamma 1 germline transcripts. In this way differentiation stage(s) of B cells committed to IgGl will be defined. In addition, T cells stimulated by the organism, particularly those stimulated by the extracellular proteins, will be further analyzed for phenotype and function. Finally, the potential role of stress proteins in regulating isotype responses physiologically will be explored. These studies will provide important new insights into the regulation of antibody class expression and the effects of the stress response in infection and immunity.

The longterm goal of this project is to elucidate the mechanism of B cell development that leads to a diverse immune repertoire. A major emphasis of this proposal is to further delineate changes in the B cell repertoire during fetal, adult, and late stages of life and how such changes are regulated. At issue is what aspects of the evolving repertoire can be explained by genetic factors, selective factors, or lineage relationships. The fetal Ig repertoire is highly restricted in terms of both V exon usage and junctional diversity. At 15 days of gestation only a handful of VH and VK exons are used repeatedly. Making use of this remarkable restriction, experiments are planned to test dominant V exons for potential genetic advantages compared with V exons that are used randomly or are underutilized. Recombination substrates will be used to measure strength of recombination signal sequences and coding end disadvantages. With the kappa locus, fetal B cells are even restricted in terms of particular VK- JK combinations, some of which recombine by inversion. Recombination substrates will be used to compare the efficiency of inversion using VK exons that are over- or under-utilized as well as to compare inversion vs deletion mechanisms. Because of the extent of restriction in V exon usage, it will be possible to examine the effects of selection on repertoire development. Two models are planned. B cells from bcl-2-Ig transgenic mice appear to be defective in apoptosis as a result of deregulated expression of bcl-2 and exhibit repertoire skewing. This will serve as a model for B cell expansion in the absence of negative selection. Bcl-2 mice with xid background will also be tested to clarify lineage relationships. Particular VK and VH exons will be followed in these mice at several stages of development. The motheaten mouse model will be used since it is likely that the defect is associated with a lack of effective CD40 ligand- CD40 interactions. Therefore, motheaten mice will be used as a prototype for lack of T-dependent positive selection leading to follicular lymphocytes. These experiments should provide important new insights into the regulation of the B cell repertoire. Understanding the generation of diversity is fundamental to questions of how the immune response works and what goes wrong in disease states such as autoimmunity.

DESCRIPTION (provided by applicant): Neurocysticercosis (NCC) is the most common parasitic disease of the central nervous system and is caused by encystment of the brain with the larval form of the cestode parasite Taenia solium. Clinical manifestations include seizures, hydrocephalus, and other symptoms associated with increased intra-cranial pressure. As the severity of the symptoms is associated with the intensity and chronicity of the local immune response, the long term goal of this project has been to characterize the immune response to NCC and the associated pathology. Apart from our previous analyses of brain specimens from NCC patients, a mouse model of NCC was developed by intra-cranial infection with larvae of the related cestode Mesocestoides corti. Preliminary findings indicate that T. solium and M. corti have teguments rich in glycoproteins and glycolipids that are released during invasion. Based upon the findings, the central hypothesis is that the uptake and storage of glycoconjugates play an important role in effecting a prolonged innate immune response, breach of the blood brain barrier (BBB), and granulomatous hypersensitivity responses. To test this, the first aims are to isolate parasitic glycoconjugates to determine if they act as persistent antigens and effect immune modulation. The approaches include use of in situ immunofluorescence to follow the amount and distribution of parasitic antigens as well as to determine their potential negative effects on antigen presentation. Another aim is to analyze in detail the innate immune response examining Toll like receptors (TLRs) and their regulation of chemokine expression. The approach is to use gene profiling to determine which genes for these molecules are regulated by infection followed by triple label immunofluorescence microscopy (IF) in situ to determine colocalisation of these molecules and the cell types producing them. In vitro cell culture assays stimulating with known TLR ligands and parasitic glycoconjugates will aid in the study of regulatory mechanisms. The last aim is to study mechanisms by which matrix metalloproteinases (MMPs) influence the integrity of the BBB. The approaches will combine gene profiling, in situ IF, and in situ zymography as above. In vitro studies and inhibitors of MMPs will elucidate mechanisms. The studies outlined will provide a deeper understanding of the immunopathology of NCC and should provide important insights for developing therapeutic interventions.

Neurocysticercosis (NCC), the most common parasitic disease of the CNS, is caused by the larval stage of Taenia solium, a cestode parasite. NCC diagnosis is generally difficult and determined by clinical criteria coupled with CT or MRI procedures. However, the utility of these diagnostic procedures in high incidence areas (developing countries of Latin America, Africa and Asia) is limited. Thus the goal of this proposal is to develop a reliable serological assay for NCC that has general utility in developing countries. The investigator proposes to develop a diagnostic ELISA assay using recombinant antigens. To this end, the specific aims of this proposal are to: 1. Identify recombinant clones of T solium glycoproteins by screening cDNA libraries with antisera directed against these proteins and express these proteins in high quantities. 2. Use the expressed proteins to develop and optimize an ELISA assay and to compare the specificity and sensitivity of the ELISA assay with that of the immunoelectrotransfer blot (EITB) assay. 3. Biochemically and molecularly characterize the glycoproteins to assess the contribution of carbohydrates to the protein's antigenicity and to map the diagnostic epitopes

[unreadable] DESCRIPTION (provided by applicant): Many infectious diseases, especially respiratory diseases, are more frequent and severe among the elderly compared with the younger population. The long-term objective of this project is to compare infection and immunity in young and aged individuals using a mouse model of pulmonary tularemia. The causative agent of tularemia is Francisella tularensis. The biotype A strain of Francisella (Schu4) has been classified as a Category A bioterrorism agent because of its ease of transmission and high mortality rate when inhaled. As such, this project is significant for the areas of both aging and bioweapons. Infection by F.t. novicia and F.t. tularensis (Schu4) will be compared in young adult and aged mice using primarily targeted macroarrays followed by in situ methodologies involving 3 and 4 color fluorescence microscopy and confocal microscopy. The aims are to perform a kinetic analysis of the infectious disease process in young and aged mice. This will allow a systematic and simultaneous analysis of the immune cells, cytokines, location of the bacteria as it disseminates, and the resulting histopathology. In addition, the protective efficacy of two protective strategies as a result of aging will be tested including the use of attenuated mutants and the use of lL-12 as an adjuvant. The underlying hypothesis is that aged animals will exhibit a delayed innate/adaptive immune response and less protection from therapeutic strategies. The proposed experiments will provide important new insights into immunosenescence, immunity to intracellular respiratory bacteria, and potential strategies should Francisella be used as a bioweapon. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Immunomicroscopy Core will consist of a state-of- the-art fluorescence imaging unit, a laser dissection microscope system, and equipment for preparing specimens including a cryostat and microtome. It will be supervised by the director, who will be responsible for training the senior research assistant and assuring its quality control. The Core will be used to define immune responses to Francisella tulerensis infection with time, as well as aid in the discovery of molecules involved in virulence such as sensitivity to intramacrophage survival and resistance to antimicrobial peptides. Analyses will be done in situ with tissues as well as at the level of single cells. As such, it will play a critical role in successfully completing the long-term goals of this Program Project: Tularemia: Pathogenesis and Host Response

DESCRIPTION (provided by applicant): The long-term goal of this project is to perform a careful kinetic analysis of the immune response to Francisella tularensis and the resulting pathology. The underlying hypothesis is that the innate immune response and/or early adaptive immune responses to pneumonic tularemia caused by F. tularensis subsp. tularensis (Schu4) is delayed or different compared to less virulent subspecies or attenuated mutants. The rationale is that comparing immune responses between virulent and less virulent strains will provide new insights into protective responses concentrating on the less virulent strains known to modulate the immune response. Three aims are proposed to compare Schu4 with targeted attenuated mutants. First, bacterial trafficking will be compared using labeled organisms followed by imaging in real time with the microPET and biodistribution of radioisotope and viable organisms. This will help determine whether differences in bacterial migration correlate with virulence as well as to identify the most important organs/tissues for study. Secondly, mice will be infected with Francisella strains/mutants (aerosol chamber) and sacrificed at various times post infection for removal of blood and relevant tissues defined in Aim 1. Initially, gene profiling will be done on isolated RNA from infected tissues by targeted macroarrays, thus focusing on immune related molecules important in the infection. Subsequently, in situ immunofluoresence approaches will be used, so that the pathogen, immune cells, and mediators can be identified at the same time. In addition, the extent of pathology will be assessed with time. Based upon our preliminary data, the third aim will concentrate on chemokines and the role of infiltrating neutrophils in disease progression. In collaboration with Project 2, we will also analyze TLR expression in situ. Information regarding virulence/protective mechanisms obtained in Projects 1-3 will interface with the 'humanized'transgenic model from Project 4. The data from this subproject, using real time imaging and in situ protocols, will provide a foundation for the disease processes associated with pneumonic tularemia. This project is therefore an integral part of the overall goal of this Program Project to understand the pathogenesis and host response to tularemia. (Cores C, B, and A required.)

DESCRIPTION (provided by applicant): Neurocysticercosis (NCC) is a common neurological disease in developing countries and the United States caused by the larva stages of the parasite Taenia solium. The pathogenesis and clinical manifestations vary with the location of the parasite within the brain and accompanying host immune response. Our long term goal has been to characterize and understand the host immune response and the pathology associated with NCC to establish better therapeutic interventions. Apart from analyses of brain specimens from NCC patients, we have developed a mouse model of NCC by intra-cranial infection with the related cestode, Mesocestoides corti. We have shown that the parasite releases glycans with distinct sugar specificities that are taken up by host cells in the CNS environment in both human and murine NCC. Of particular relevance, two C-type lectin receptors (CLRs) are highly up-regulated after parasite infection: mannose receptor (MRC1) and macrophage galactose C-type lectin (MGL1) which recognize sugars from parasite and host origin respectively. We hypothesize that recognition of parasite- released glycans via MRC1 leads to protective CNS responses. This is based on the observation that Mrc1-/- mice survive significantly longer and exhibit reduced CNS pathology that correlates with increased infiltration a novel population of myeloid cells with a suppressor phenotype. In contrast, Mgl1-/- mice show decreased survival and up-regulation of inflammatory cytokines. We further hypothesize that CLRs differentially modulate myeloid plasticity thus regulating the degree of inflammation vs. suppression in murine NCC. To test this, we will identify the effects of MRC1 and MGL1 in myeloid cell function during the early and late stages of murine NCC (Aim 1), and determine the mechanisms by which MRC1 modulate effector antigen specific T cell immune responses (Aim 2). Our proposed studies will provide the first detailed assessment of CLR expression and function in CNS parasite infections and present novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Neurocysticercosis is the most frequent parasitic disease affecting the central nervous system (CNS), and the clinical symptoms can be devastating and lifelong. The severity of the symptoms is associated with the intensity of the immunological response and involves innate immune receptors called C-type lectins, understudied molecules in the CNS. C-type lectins can induce immune suppression so understanding their function will likely result in new therapeutic strategies.