Medical Sciences Commons^™

Isolation And Immunological Characterization Of A Group Of New B Lymphomas From Cba Mice, Venkatachalam Udhayakumar, Peter H. Brodeur, M. S. Rajagopalan, Stephen Zimmer, Karen E. Pollok, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

We have isolated and characterized a new series of B lymphomas which occurred spontaneously in a group of CBA N mice that were transferred with spleen or lymph node cells from 24-month-old CBA Ca mice. Tumor cell lines from six CBA N mice that received spleen cells were rescued and designated as BKS-2, BKS-3, BKS-4, BKS-5, BKS-6, and BKS-7. Also, tumor cells from a recipient of lymph node cells were rescued and the resulting cell line was designated BKL. These tumor cells expressed membrane immunoglobulin (μ,κ), major histocompatibility complex Class I and Class II molecules, B220, Lyb8, Fc receptors, J11d, …

A Specific Defect In The Proliferative Capacity Of B Cells From Old Mice Stimulated With Autoreactive T Cells, Prakash S. Nagarkatti, Aruna Seth, Mitzi Nagarkatti, Natarajan Muthusamy, Barbara Rychlik, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B …

Camp Antagonizes Interleukin 2-Promoted T-Cell Cycle Progression At A Discrete Point In Early G1., Kirk W. Johnson, Bruce H. Davis, Kendall A. Smith

Dartmouth Scholarship

T lymphocytes are stimulated to proliferate in an autocrine/paracrine manner by the lymphokine interleukin 2 (IL-2). In seeking further insight into the mechanisms by which IL-2 induces progression of T cells through the G1 phase of the cell cycle, studies were performed with agents that increase cellular adenosine 3',5'-cyclic monophosphate (cAMP), a well-known inhibitor of lymphocyte growth. The addition of dibutyryl-cAMP, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine to an IL-2-dependent murine T-cell line evoked a dose-related suppression of S-phase transition without affecting cellular viability. Moreover, elevation of cAMP levels led to an accumulation of uniformly small cells, suggesting an arrest in …

Determination Of Antibody Levels In Periapical Lesions Against Sixteen Oral Microorganisms By The Elisa Technique, Sammee Lee Jones

Loma Linda University Electronic Theses, Dissertations & Projects

The root canal system, which includes the periapical region of the tooth, may become infected by several types of microorganisms which are normally associated with these tissues. A bacteria-specific antibody response may occur at the local level, where various classes of immunoglobulins have been found and reported in the literature. The purpose of this study was to quantitatively measure locally produced immunoglobulin' levels in periapical lesion abscess materials and to determine the specific reactivity of these antibodies to several microorganisms which are normal flora, as well as being previously associated with the development and pathogenesis of endodontic infections. Periapical lesions …

Properties Of Anti-Lyb-2-Mediated B-Cell Activation And The Relationship Between Lyb-2 Molecules And Receptors For B-Cell Stimulatory Factor-1 On Murine B Lymphocytes, Subbarao Bondada, Joan Morris, A. R. Baluyut

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to …

Impaired Autoreactive T Cell-Induced T Cell-T Cell Interaction In Aged Mice, Venkatachalam Udhayakumar, Subbarao Bondada, Aruna Seth, Mitzi Nagarkatti, Prakash S. Nagarkatti

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Self-Ia-reactive (autoreactive) L3T4⁺ T cell clones have been shown earlier to stimulate the proliferation of syngeneic naive L3T4⁺ T cells and initiate a T cell-T cell (T-T) interaction leading to the generation of immunoregulatory circuits. Since aging has been shown to be associated with a decline of the immune responsiveness, age-related alterations in the T-T interaction was investigated in the present study. Using several I-E^d-specific autoreactive T cell clones isolated from 2- to 3-month-old (young) DBA/2 mice as stimulators, it was observed that L3T4⁺ T cells from 22- to 24-month-old (aged) DBA/2 mice, failed to …

Early Events In B-Cell Activation: Anti-Lyb2, But Not Bsf-1, Induces A Phosphatidylinositol Response In Murine B Cells, Stephan A. Grupp, Judith A. K. Harmony, Arthur R. Baluyut, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent …

Role Of Serum Amyloid P Component In Immune Clearance, Cindy L. Bristow

MUSC Theses and Dissertations

In order to clarify the mechanism of interaction of serum amyloid P component (SAP) with complement, the interaction of SAP with Clq was studied. It is known that SAP binds Sepharose 4B in the presence of calcium. 125I-Clq was retained on the Sepharose when purified 125I-Clq was incubated with SAP prior to affinity chromatography on Sepharose. In the absence of SAP, the 125I-Clq was not retained. To further examine the interaction of SAP with Clq, SAP was incubated at varying ratios with Clq. These mixtures were examined via crossed immunoelectro-immunoelectrophoresis against goat anti-SAP. A change in the electrophoretic behavior of …

Interleukin 2 Regulates Its Own Receptors., Kendall A. Smith, Doreen A. Cantrell

Dartmouth Scholarship

The cell surface density of high-affinity membrane receptors for the T-lymphocytotrophic hormone interleukin 2 (IL-2) determines the rate of T-cell-cycle progression. Since 10-fold greater numbers of IL-2 receptor molecules were found by using a radiolabeled monoclonal antibody reactive with IL-2 receptors (anti-Tac) compared with binding of IL-2, the functional relationship of the binding sites recognized by both of these ligands was assessed. In the presence of cycloheximide, IL-2 binding sites declined with a half-time (t1/2) of 2.6 hr, whereas the decay of anti-Tac binding sites was much slower (t 1/2 = 6.4 hr). Moreover, after limited membrane proteolysis, the half-time …

Triggering Of The T3-Ti Antigen-Receptor Complex Results In Clonal T-Cell Proliferation Through An Interleukin 2-Dependent Autocrine Pathway., Stefan C. Meuer, Rebecca E. Hussey, Doreen A. Cantrell, James C. Hodgdon, Stuart F. Schlossman, Kendall A. Smith, Ellis L. Reinherz

Dartmouth Scholarship

Human T-cell clones and anti-T-cell-receptor antibodies (clonotypic) directed at surface receptors for antigen (T3-Ti molecular complex) as well as anti-interleukin 2 (IL-2) and anti-IL-2-receptor antibodies were utilized to investigate the mechanism by which alloantigens or antigen plus self-major histocompatibility complex (MHC) (i.e., physiologic ligand) trigger specific clonal proliferation. Soluble or Sepharose-bound anti-Ti monoclonal antibodies, like physiologic ligand, enhanced proliferative responses to purified IL-2 by inducing a 6-fold increase in surface IL-2 receptor expression. In contrast, only Sepharose-bound anti-Ti or physiologic ligand triggered endogenous clonal IL-2 production and resulted in subsequent proliferation. The latter was blocked by antibodies directed at either …

Activation Of B Lymphocytes By Monovalent Anti-Lyb-2 Antibodies, Subbarao Bondada, D. E. Mosier

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Small resting B lymphocytes are activated when their cell surface immuno-globulin (Ig) receptors interact with specific antigen or anti-Ig antibodies. Such a receptor-ligand interaction leads to enlargement and proliferation of B lymphocytes. These activated B cells can subsequently mature into Ig-secreting cells in the presence of appropriate accessory cell signals (1-3). However, monovalent anti-Ig antibodies cannot induce B cell activation even though they can bind to the surface Ig (4-7). It has thus been concluded that membrane Ig has to be cross-linked to induce early steps in B cell activation (membrane depolarization; 8) as well as later steps (proliferation). T …

Biochemical Characterization Of Human Thy 1, Lynda F. Bonewald

MUSC Theses and Dissertations

The present studies were undertaken in order to a) verify the presence of a human Thy 1 antigen, b) demonstrate its similarity to murine Thy 1, and c) biochemically characterize the molecule. Previously, a 25,000 molecular weight molecule had been isolated from a human T lymphoblastoid cell line, Molt 3, using isolation procedures most frequently used for the isolation of rodent Thy 1. The human Thy 1 (p25) was demonstrated to be biochemically similar to murine Thy 1.2 by molecular weight and ability to bind Lens Culinaris lentil lectin. Recently, we confirmed similarity between the murine and the human molecule …

The Utilization Of Response Surface Techniques In Survival Experiments, Leonard Edward Buchner

MUSC Theses and Dissertations

Linear regression procedures are described which give an unbiased estimate of the percent functional viability of rat polymorphonuclear leukocytes (PMN's). These statistical techniques allow discrimination between variables which result in increased PMN survival after freezing. A review of current response surface techniques was made as they relate to optimizing the functional viability of PMN's after freezing. Experimental designs based on the steepest ascent and simplex methodologies were considered. Since the hill climbing efficiency of the steepest ascent and simplex methods are comparable, the steepest ascent procedure was selected as it is more readily adaptable to a batch mode analysis.

Proteins Antigenically Related To The Human Erythrocyte Glucose Transporter In Normal And Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts., Donald W. Salter, Stephen A. Baldwin, Gustav E. Lienhard, Michael J. Weber

Dartmouth Scholarship

Antibody raised against the purified human erythrocyte glucose transporter specifically precipitated four proteins from normal and Rous sarcoma virus-transformed chicken embryo cells: a major protein of Mr 41,000 and minor proteins of Mr 68,000, 73,000, and 82,000. The Mr 41,000 and 82,000 proteins were found only in a membrane fraction, not in the soluble fraction, and displayed a heterogeneous mobility on NaDodSO4/polyacrylamide gel electrophoresis, suggesting glycosylation. The Mr 41,000 and 82,000 proteins were increased in amount after malignant transformation in direct proportion to the increase in hexose transport rate, and the increase was dependent on the expression of the src …

T Cell Growth Factor Receptors. Quantitation, Specificity, And Biological Relevance, Richard J. Robb, Allen Munck, Kendall A. Smith

Dartmouth Scholarship

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain …

Rat Dendritic Cells Function As Accessory Cells And Control The Production Of A Soluble Factor Required For Mitogenic Responses Of T Lymphocytes., Wolfgang E. Klinkert, Jon H. Labadie, James P. O'Brien, Carl F. Beyer, William E. Bowers

Dartmouth Scholarship

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred …

Cell Mediated Immunity By Cytotoxicity Assay And The Effect Of Corynebacterium Parvum And Radiation On Mice Bearing Herpes-Induced Tumors, Robert B. Stagg

Loma Linda University Electronic Theses, Dissertations & Projects

Cell mediated immune (CMI) cytotoxic reactivity of Balb/c mice against H238 cells, a Herpes simplex virus (HSV) Type 2 - induced sarcoma, was measured by the ¹²⁵IUdR release assay. The Balb/c mouse response to the growing HSV-induced tumor, treated with radiation and , was measured by survival rate, tumor growth and immune cytotoxicity of spleen and peritoneal exudate cells (PEC) as determined by ¹²⁵IUdR release assay. Subcutaneous (s.c.) inoculation of 1 x 10⁶ H238 cells (high dose) produced progressive tumor growth while s.c. inoculation of 1 x 10⁴ H238 cells (low dose) produced no tumors. A …

Role Of A Nonimmunoglobulin Cell Surface Determinant In The Activation Of B Lymphocytes By Thymus-Independent Antigens*, Subbarao Bondada, Donald E. Mosier, Aftab Ahmed, James J. Mond, Irwin Scher, William E. Paul

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Lyb 5 is a B-cell alloantigen which is expressed on 50-60% of B cells. It was defined originally on the basis of cytotoxicity. We have described a new reactivity within the anti-Lyb 5 serum on the basis of selective inhibition of antibody responses in vitro by this antiserum in the absence of complement. This inhibitory activity of anti-Lyb 5.1 serum appears to be due to recognition of antigenic determinants different from the prototype antigens detected in the cytotoxicity assay. Anti-Lyb 5 serum incorporated into spleen cell cultures selectively inhibits antibody responses to a class of thymus-independent antigens (TI-2) previously characterized …

Tritium (3h) Radiolabeling Of Protein A And Antibody To High Specific Activity: Application To Cell Surface Antigen Radioimmunoassays, Ronald L. Wilder, Catherine C. Yuen, Subbarao Bondada, Virgil L. Woods, Cornelius B. Alexander, Rose G. Mage

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Staphylococcal protein A and several different immunoglobulins have been radio-labeled to high specific activities (> 106 cpm/pg) by reductive methylation with tritiated (3H) sodium borohydride. The proteins retain excellent functional and antigenic properties. The utility of these reagents in a variety of assays for cell surface antigens is illustrated. The results indicate that this radiolabeling procedure may become the method of choice for many cell surface and solution immunoassays.

The Pi System Of Alpha-L-Protease Inhibitor (Alpha-L-Antitrypsin) Biochemical Immunological And Genetic Studies In Health And Disease, Philippe Arnaud

MUSC Theses and Dissertations

The results of our personal studies of alpha-l-protease inhibitor (APi, formerly termed alpha-l-antitrypsin), the major inhibitor of protease in extracellular fluids, are presented in this dissertation. In the first chapter, we describe techniques used for APi quantitation and determination of Pi phenotypes. After a brief review of the available techniques, we report several improvements on our original technique of Pi phenotyping using thin-layer isoelectric focusing. Particular attention is given to biochemical and immunological methods for verification of the results obtained by isoelectric focusing. Three complementary techniques are reported: protease probe, crossed immunoelectrophoresis, and print immunofixation. Analysis of the results from …

Inactivation Of Brain Hexokinase By An Adenosine 5'-Triphosphate Analog, Subbarao Bondada, Umakant W. Kenkare

Microbiology, Immunology, and Molecular Genetics Faculty Publications

An analog of ATP, 6-mercapto-9+nribofuranosylpurine 5’-triphosphate (SH-TP), functions as a phosphoryl donor for the reaction catalyzed by hexokinase. Incubation of the enzyme with millimolar concentrations of this reagent led to its rapid inactivation. However, the corresponding monophosphate derivative of this reagent was much less effective. A plot of the initial rate of inactivation versus the concentration of SH-TP exhibited saturation, suggesting the formation of a reversible complex between the enzyme and SH-TP prior to the inactivation reaction. The dissociation constant of this enzyme-inhibitor complex was the same as the K, of this reagent with respect to hexokinase in the phosphoryl …

Reaction Of Brain Hexokinase With Tetranitromethane: Oxidation Of Essential Thiol Groups, Subbarao Bondada, Umakant W. Kenkare

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Treatment of bovine brain mitochondrial hexokinase with a fivefold molar excess of tetranitromethane (TNM) at pH 8.0 results in a complete loss of activity of the enzyme. Spectral measurements and amino acid analysis showed that only cysteinyl residues were modified by TNM under these conditions. The products of the reaction of TNM with the enzyme were cysteic acid and disulfides as found by amino acid analysis of the carboxymethylated hexokinase. Failure of 2-mercaptoethanol to reverse the effect of TNM suggested that the thiol group that is oxidized to cysteic acid is the one essential for enzyme activity. Substrates glucose and …

A Batch Method For The Isolation Of Immunogenically Pure Igg, Iga, And Igm From Human Sera, Samuel Christopher Powell

Chemistry & Biochemistry Theses & Dissertations

A method is described for preparing an enriched fraction of the three major antibodies, immunoglobulins IgG, IgA and IgM, from normal human serum by a batch method. Selective precipitation of these proteins was carried out in successive steps using polyethylene glycol (PEG) of average molecular weight 6,000 at concentrations of 7%, 12% and 14% (16) . Zinc sulfate (10 mM) was used to remove highly immunogenic alpha₂ macroglobulin from the IgM fraction. IgA, IgM and IgG were further purified by adsorption methods using DEAE-cellulose. Quantitation and qualitation of the enriched fractions showed nearly complete separation of each of the …

Lethatlity Of And Elicitation Of Protective Antibody By Vibrio Parahaemolyticus Attc 17803, Richard Carlucci

Masters Theses & Specialist Projects

The LD₅₀ of Vibrio parahaemolyticus ATCC 17803 for 13- to 18-g male mice, strain ICR, as determined by the 50% endpoint method, was found to be an estimated 7 x 10⁷ CFU, when administered intraperitoneally in 0.5 ml of 24-h broth culture, O. D. 0.5 at 650 nm, diluted to 10^-0.72.

The elicitation of protective antibody by this bacterium in male New Zealand White rabbits was demonstrated by testing control, baseline, and hyperimmune rabbit sera for their protective effect against 2 LD₅₀ of the bacterium administered intraperitoneally in 13- to 18-g male mice, strain ICR. …

Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses, Catherine M. Crosby, William E. Matchett, Stephanie S. Anguiano-Zarate, Christopher A. Parks, Eric A. Weaver, Larry R. Pease, Richard J. Webby, Michael A. Barry

Nebraska Center for Virology: Faculty Publications

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed “single-cycle” adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors …