Kenneth J. Blank

Mechanisms of Caloric Restriction Affecting Aging and Diseasea

Caloric restriction (CR) appears to affect aging by the inhibition of the specific chronic diseases which occur at increasing frequency with age. A common disease in F-344 rats, granulocytic leukemia, appears to have a window where it is sensitive to the effects of CR. Other diseases, such as pituitary adenomas, appear to have a different relationship to growth in the animal. Additionally, a model for the major disease for a number of long-lived strains of mice, lymphoma, which CR effects by inhibiting the expression of the causative agent, is being developed. Evaluation of the effects of CR on neoplasia, degenerative disease and physiological parameters suggests that the major factors in expression of these diseases is the alteration of growth factors, hormonal status, etc., and that these alterations also affect strain-specific pathologies depending on when they are changed in the life span. Effecting different diseases at different times in the life span, long-term CR, by limiting exposure to endogenous growth factors, altering physiological characteristics, and limiting exposure to food toxicants, inhibits the onset of disease, and its sequela, aging.

Methylxanthine-induced inhibition of the antigen- and superantigen-specific activation of T and B lymphocytes.

Methylxanthines have been shown to have a variety of effects on hematopoietic cell activation and function. These compounds inhibit cAMP-specific phosphodiesterase activity resulting in increased levels of intracellular cAMP. In the present study, we examined the effects of two methylxanthines, pentoxifylline (PTX) and caffeine, on the responses of both mouse and human lymphocytes to stimulation with polyclonal T- and B-cell mitogens, antigens, and the microbial superantigen, staphylococcal enterotoxin B (SEB). Both PTX and caffeine significantly inhibited mitogen- and SEB-induced proliferation by murine spleen cells, SEB- and antigen-induced proliferation and lymphokine secretion by murine Th1 and Th2 clones, and the generation of antigen-specific antibody producing murine spleen cells. These compounds also inhibited the proliferative responses of human lymphocytes to phytohemagglutinin, SEB, and tetanus toxoid. Efforts to determine whether these methylxanthine compounds mediated their inhibitory effects through a specific protein kinase pathway revealed a role for cAMP-dependent protein kinase A in methylxanthine-induced immunomodulation. However, it is possible that a protein kinase A-independent pathway may also be involved. These data demonstrate that the methylxanthines, PTX and caffeine, have profound effects on cells of the immune system and may have a potential use as immunotherapeutic agents in the treatment of various inflammatory conditions and autoimmune diseases.

Strain-dependent migration of lymphocytes to the vaginal mucosa after peripheral immunization.

Abstract We have previously demonstrated a genetic predisposition among mice regarding their ability to be protected against vaginal candidiasis after peripheral immunization. Both BALB/c and (BALB/c× C57BL/6) F1 mice are protected against vaginal candidiasis after subcutaneous immunization with Candida albicans extract and C57BL/6 mice are not protected by this immunization. In the present study, the ability of F1-derived immune cells to transfer protection to naive parental strains was observed in BALB/c recipient mice, but not apparent in B6 recipient mice. This result is highly suggestive that the microenvironment of the B6 mouse is responsible for the susceptible phenotype. Genetic studies using (BALB/c× C57BL/6)F1× C57BL/6 backcross mice demonstrated that two genes appeared to regulate the protective effect of peripheral immunization to vaginal challenge. Microsatellite mapping indicated that candidate loci involved in controlling the immune response to vaginal candidiasis after peripheral immunization included the intercellular adhesion molecule-1 (ICAM-1), the Icam-1 related sequence 1, and the Fc epsilon RII (P<0.01). Thus, the ability of cells to bind to vaginal endothelial cells may play an important role in protection against vaginal candidiasis mediated by peripheral immunization.

Rational design of cytotoxic T-cell inhibitors.

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 α-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analogs activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.

Pentoxifylline- and caffeine-induced modulation of major histocompatibility complex class I expression on murine tumor cell lines

The methylxanthines, pentoxifylline (PTX) and caffeine, modulated major histocompatibility complex class I expression on three constitutively class I-positive murine T cell lymphoma lines. On two cell lines, PTX or caffeine treatment enhanced H-2K and H-2D expression. Treatment with PTX and either interferon-gamma, interferon-alpha/beta, tumor necrosis factor, or lymphotoxin increased the levels of K and D expression above those observed following treatment with either PTX or cytokines alone. On the third cell line, PTX or caffeine treatment enhanced D expression and reduced K expression. Treatment with PTX and any of the cytokines resulted in a level of D expression greater than that seen following treatment with either PTX or cytokines alone. However, PTX inhibited the cytokine-induced enhancement of K expression. PTX and caffeine did not induce class I expression on three constitutively class I-negative murine T cell lymphoma lines. Dibutyryl cAMP modulated class I expression in the same manner as PTX and caffeine. The PTX- and caffeine-mediated enhancement of class I expression was at least partially blocked by an inhibitor of cAMP-dependent protein kinase A. These results demonstrate that PTX and caffeine are able to regulate class I expression and that this regulation involves a cAMP-dependent mechanism.

Superantigens and Microbial Pathogenesis

The results of a randomized, controlled trial of endoscopic ligation of esophageal varices compared with sclerotherapy by Laine and colleagues, in this issue of Annals, is the second report of a relatively new endoscopic technique. The two techniques appear to be approximately comparable, except that the complication rate is less with ligation. Whether ligation will replace sclerotherapy remains to be seen. Recent analysis of various bacterial and viral products has revealed that microbial proteins may be not only responsible for microbial infection and disease but also connected to the onset of autoimmunity. These microbial products are called superantigens because of their strong effect on the proliferation of certain T-lymphocyte populations. Superantigens elicit various biological activities, including lymphocyte mitogenesis, pyrogenicity, depressed antibody production, and shock [1, 2]. We describe the effects of microbial superantigens on immune function and their possible association with various acute and chronic inflammatory diseases. T cells mediate protection against infectious agents by producing lymphokines after activation by specific antigens. Lymphokines affect the function of other cell types, including phagocytic cells, involved in antimicrobial immunity. Most mature T cells express receptors (T-cell receptors) composed of and chains, which are necessary for the specific recognition of antigens [3]. Both and chains consist of constant and variable (V) regions. Many V and V families of genes can encode the V regions of T-cell receptors; however, the antigen receptor of a single T cell is encoded by only a single V and V family member. T-cell receptors only recognize antigens when the antigens are bound in the groove of major histocompatibility complex class II molecules. These class II molecules are expressed on the surface of antigen-presenting cells such as macrophages. The class II-antigen peptide complex on antigen-presenting cells is recognized by that portion of the T-cell receptor formed by interaction between the V and V chains (in much the same way that antigen binds to the variable regions of light and heavy chains of antibody). This binding process results in the antigen-specific activation of only the small number of T cells bearing the specific receptor for that particular class II-antigen peptide complex. In contrast to antigens, superantigens bind only to the V chain and induce activation of the large number of cells expressing specific V chains. The T-cell response to superantigens is therefore much larger than antigen-specific responses. Like antigens, superantigens require antigen-presenting cells expressing class II molecules to induce T-cell proliferation; however, superantigens do not bind to class II molecules through the groove structure [1, 2, 4, 5]. Like superantigens, mitogens such as concanavalin A and phytohemagglutinin stimulate large numbers of T cells, but, unlike superantigens, mitogens do so without regard for the particular V chain expressed by the T cells and do not have to be bound to class II molecules to induce T-cell activation. There is increasing evidence of a fundamental difference between the effect of in vitro and in vivo exposure of T cells to superantigens. In vitro, superantigens cause a dramatic proliferation of T cells bearing specific V molecules. Various superantigens have been reported to stimulate the production of several cytokines, including interleukin-2, 4,and 6,tumor necrosis factor- and , and interferon- [6-9]. On the other hand, exposure to superantigen in vivo can abrogate the T-cell response to subsequent stimulation with the same superantigen in vitro [10]. Activation by superantigen in vivo, therefore, can result in either the establishment of unresponsiveness or the deletion of T cells. Staphylococcus aureus produces a family of protein enterotoxins that possess superantigen properties [11]. These enterotoxins belong to a larger family of related gram-positive bacterial pyrogenic toxins including toxic shock syndrome toxin 1 (TSST-1). A Mycoplasma arthritidis-secreted protein [12] and streptococcal M protein [13] also possess superantigen properties. Several toxic syndromes are associated with staphylococcal enterotoxins and other bacterial superantigens. Although staphylococcal enterotoxins are generally associated with food-borne intoxication, they have also been implicated in the toxic shock syndrome [14]. The superantigens TSST-1 and staphylococcal enterotoxins induce a toxic shock syndrome-like disease in a rabbit model of menstrual toxic shock syndrome [15]. Although TSST-1 has been strongly associated with menstrual toxic shock syndrome, other staphylococcal toxins, primarily staphylococcal enterotoxin B, play a pathogenetic role in cases not associated with menstruation. Recent studies have shown that patients with the toxic shock syndrome have a predominance of peripheral T cells expressing V 2 chains, a subset of the V family [16]. These results suggest that superantigen-induced activation of T cells expressing specific V chains may result in massive lymphokine production that might, in turn, be responsible for the clinical syndrome observed. From these studies, it is clear that not all in vivo exposure to superantigen leads to T-cell unresponsiveness as described above. The circumstances under which unresponsiveness or disease is induced by superantigens are being investigated. Other bacterial superantigens have also been implicated in the development of various disease states. The identification of M protein of group A streptococci as a superantigen and its interaction with human T cells may be of central importance in the pathogenesis of poststreptococcal autoimmune diseases, acute rheumatic fever, and acute glomerulonephritis [13]. Mycoplasma arthritidis, predominantly a rodent pathogen, can induce a chronic inflammatory arthritis in rats and has been cultured from the bone marrow of patients with systemic lupus erythematosus [17, 18]. It has been postulated that the M. arthritidis superantigen-induced T-cell activation may contribute to the polyclonal B-cell activation seen in patients with systemic lupus erythematosus. In addition, the finding that V () 14-positive T cells are significantly elevated in the synovial fluid of affected joints in patients with rheumatoid arthritis when compared with peripheral blood from such patients suggests that the development of disease may initially involve the superantigen-induced activation of V 14-bearing T cells [19]. Superantigens have also been found to be produced by retroviruses [20, 21]. The in vivo expression of one of these retroviruses, mouse mammary tumor virus, results in the selective depletion of V 14-bearing T cells [22]. A selective depletion of T cells that express specific V chains also occurs in patients infected with human immunodeficiency virus (HIV), suggesting that an HIV superantigen exists [23]. The role of this superantigen in the pathogenesis of HIV-related disease is being investigated. As described above, activation by superantigen in vivo can, under certain circumstances, either establish unresponsiveness in T cells expressing specific V chains or cause their deletion. Although the mechanism of this unresponsiveness remains poorly understood, it may be possible to selectively disable T cells bearing certain V molecules that are involved in autoimmune disorders. Thus, superantigens may have a potential use in prophylaxis or treatment of certain autoimmune disorders. This possibility is shown by the fact that the in vivo administration of a superantigen, Staphylococcus enterotoxin E, but not other Staphylococcus enterotoxins, ablated the induction of experimental allergic encephalomyelitis, a disease resembling multiple sclerosis [24]. Thus, superantigen-mediated induction of T-cell nonresponsiveness may have relevance not only for the role of these molecules in microbial pathogenesis but also for therapeutic considerations in autoimmune diseases involving specific V-bearing T cells in their pathogenesis. However, the systemic administration of these products would not be without risk; the intravenous administration of these toxins in mice resulted in several toxic side effects. Molecular alterations of superantigens into less toxic forms that retain their activity on T cells in vivo may result in effective agents for treatment of autoimmune disorders. Bacterial superantigens may have a role in the induction and development of autoimmunity as well as acute and chronic inflammatory disease states. The polyclonal expansion of superantigen-activated T cells may result in the production of large quantities of lymphokines and the activation of autoreactive lymphocyte populations. Superantigens have been proposed to be relevant to the induction of rheumatoid arthritis and multiple sclerosis because of the expansion of particular autoreactive V-bearing T cells in these patients. However, the role of these expanded T-cell populations in disease developments needs further evaluation. In addition, the nature of these bacterial products and their inhibitory effects on specific T cells expressing particular V chains suggest that these proteins may have potential as therapeutic agents in the treatment of T-cell-mediated autoimmune and inflammatory disease induction and progression.

Differences between young and aged mice in suceptibility to Friend virus

Friend virus (FV) is a murine leukemia virus that infects progenitor red blood cells and causes an erythroleukemia in susceptible mouse strains, resulting in splenomegaly. Several genetic loci of the host have been identified that affect erythroleukemia development, differentiation status of target cells and virus replication. Since age may change expression of these loci, age may affect FV disease. To explore this possibility, FV expression in four genetically diverse strains of mice of different ages was examined. Extent of viral replication and of disease were evaluated by measuring spleen focus forming units (SFFU), spleen weight and reverse transcriptase (RT) activity in target organs. Young DBA/2 and (C57BL/6 x DBA/2)F1 mice exhibited a greater level of virus expression than their aged counterparts in all parameters investigated. Young CBA/Ca mice had slightly higher spleen weights and SFFU values than aged CBA/Ca mice, but a definitive age-related change was not observed in the RT activity of the target organs. C57BL/6 mice, which are genetically resistant to the development of FV-induced erythroleukemia, exhibited a limited degree of virus replication that was not effected by the age of the animal. Our results indicate that the age of the mouse, as well as the genetic background, can contribute to the level of susceptibility to FV.

Introduction of the H-2Dk gene into a class I-negative tumor cell line confers interferon-γ inducibility upon the silent endogenous H-2Kk gene

Kgv cells do not constitutively express class I mRNA or protein. Interferon (IFN)-gamma, but not IFN-alpha/beta, induces H-2Dk expression. IFN does not induce H-2Kk expression. We examined constitutive and IFN-inducible class I expression on Kgv cells stably transfected with genomic clones of H-2Kk or H-2Dk and on somatic cell hybrid lines constructed between Kgv cells and constitutively class I-positive cells of a distinguishable H-2 haplotype. Our results suggest that both the lack of constitutive class I expression and the inability of IFN-alpha/beta to induce class I expression on Kgv cells are primarily due to cis-regulatory mechanisms. However, stable introduction of the H-2Dk gene into Kgv cells conferred IFN-gamma inducibility upon the silent endogenous H-2Kk gene. Therefore, the failure of IFN-gamma to induce H-2Kk expression on Kgv cells is due, at least in part, to a trans-regulatory mechanism.