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Biomedical subjects

N H Ruddle

Publications and source records attributed to N H Ruddle.

At least 55 records · Page 3Linked to original sources
PubMed1993

T helper 1 (Th1) functional phenotype of human myelin basic protein-specific T lymphocytes.

Multiple sclerosis (MS) is widely accepted as an autoimmune disease with myelin basic protein (MBP) a candidate autoantigen. In the current report, human T cell lines specific for an immunodominant region of MBP were shown to have a functional phenotype similar to T helper 1 (Th1) inflammatory cells of the mouse on the basis of their antigen-specific cytotoxic activity and production of interferon-gamma and lymphotoxin/tumor necrosis factor-alpha, but not interleukin-4. In experimental allergic encephalomyelitis (EAE), a proposed animal model for MS, MBP-specific T cell lines which mediate disease are of the Th1 subtype. Thus, MBP-specific T cells in humans exist which are phenotypically similar to MBP-specific encephalitogenic T cells in murine EAE.

Adult↗
PubMed1992

Insulitis in transgenic mice expressing tumor necrosis factor beta (lymphotoxin) in the pancreas.

Tumor necrosis factor beta (TNF-beta) (lymphotoxin) may play an important role in the immune response and pathologic inflammatory diseases. Insulitis is an important early step in the development of insulin-dependent diabetes mellitus. To understand better the role of TNF-beta in the regulation of inflammation and type 1 diabetes, we produced transgenic mice in which the murine TNF-beta gene was regulated by the rat insulin II promoter. The transgene was expressed in the pancreas, kidney, and skin of transgenic mice. The expression of TNF-beta in the pancreas of transgenic mice resulted in a leukocytic inflammatory infiltrate consisting primarily of B220+ IgM+ B cells and CD4+ and CD8+ T cells. The insulitis is reminiscent of the early stages of diabetes, though the mice did not progress to diabetes.

Animals↗
PubMed1992

Molecular regulation of tumor necrosis factor-alpha and lymphotoxin production in T cells. Inhibition by prostaglandin E2.

The effects of prostaglandin E2 (PGE2) and other eicosanoids were determined on tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin (LT; TNF-beta) production by murine Th1, antigen-specific major histocompatibility complex class II-restricted T cell clones. T cells activated with immobilized anti-CD3 or with soluble concanavalin A (ConA) produced approximately 100-1,000 units/ml TNF-alpha/LT as determined by cytotoxicity against the WEHI-164 murine fibrosarcoma cell line. TNF-alpha/LT biological activity was induced rapidly with significant production evident approximately 4 h after stimulation with either anti-CD3 or ConA. PGE2 inhibited the anti-CD3- and ConA-induced production of TNF-alpha/LT bioactivity in a dose-dependent manner. Incubation with 1 x 10(-9) M PGE2 inhibited production of TNF-alpha/LT biological activity from anti-CD-activated F1.28, or 450A.1 T cell clones by approximately 50%. Incubation with 1 x 10(-7) M PGE2 resulted in 90% inhibition of biological activity. PGE2 also inhibited both TNF-alpha and LT mRNA accumulation by more than 90%. These results suggest that the reduced production of cytotoxic activity in the presence of PGE2 was caused by the inhibition of both TNF-alpha and LT. No inhibition of T cell TNF-alpha or LT production was observed when anti-CD3- or ConA-activated cells were incubated with PGD2, PGF2 alpha, 5-hydroxyeicosatetraenoic acid or leukotriene C4. Nuclear run-on experiments indicated that the PGE2-mediated decrease of TNF-alpha and LT mRNA accumulation was caused, in part, by an inhibitory effect on the transcription of these genes. This is the first report of PGE2-mediated inhibition of TNF-alpha in T cells and PGE2-mediated inhibition of LT production in any cell type.

Animals↗
PubMed1992

Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta).

Distinctions between tumor necrosis factor, TNF-alpha, and lymphotoxin, TNF-beta, have previously been based on the differences between their protein sequences, biological activity, and molecular regulation. In the past year, elucidation of the molecular nature of the two molecules and the interactions with common receptors has emphasized their similarities, although profound differences continue to emerge with regard to their mode of production, transcription rates, mRNA half-lives, and the importance of various DNA regulatory sequences. A role for both TNF-alpha and TNF-beta has recently been suggested with regard to disease, particularly multiple sclerosis. The past year has also seen the description of an extensive microsatellite polymorphic system which should provide a more definitive understanding of the association of the TNF locus with disease.

Animals↗
PubMed1992

A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation.

Lymphotoxin (LT; also known as tumor necrosis factor-beta) is a pleiotropic cytokine whose expression is tightly regulated in most cells and is repressed prior to activation signals. In some early B cells and Abelson murine leukemia virus-transformed pre-B-cell lines, LT mRNA is constitutively expressed. To examine the molecular regulation of the LT gene in a constitutively expressing cell line, we studied the Abelson murine leukemia virus-transformed lines PD and PD31. As demonstrated by primer extension analysis, constitutively expressed pre-B-cell-derived and inducibly expressed T-cell-derived LT mRNA were initiated at the same cap sites and predominant cap site utilization was conserved. Furthermore, we delineated an upstream activating sequence that was an important functional component of lymphotoxin transcriptional activation in PD and PD31 cells. The upstream activating sequence was localized to an essentially homopolymeric A + T-rich region (LT-612/-580), which was bound specifically by recombinant human high-mobility group I protein (HMG-I) and a PD/PD31 nuclear extract HMG-I (HMG-I-like) protein. The nuclear extract-derived HMG-I-like protein was recognized by anti-HMG-I antibody and bound to LT DNA to effect an electrophoretic mobility shift identical to that of bound recombinant human HMG-I. These findings implicate HMG-I in the regulation of constitutive lymphotoxin gene expression in PD and PD31 cells. HMG-I and HMG-I-like proteins could facilitate the formation of active initiation complexes by altering chromatin structure and/or by creating recognition sites for other activator DNA-binding proteins, some of which may be unique to or uniquely modified in these constitutive LT mRNA producers.

Base Sequence↗
PubMed1990

An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis.

Uncertainty regarding pathogenic mechanisms has been a major impediment to effective prevention and treatment for human neurologic diseases such as multiple sclerosis, tropical spastic paraparesis, and AIDS demyelinating disease. Here, we implicate lymphotoxin (LT) (tumor necrosis factor beta [TNF-beta]) and TNF-alpha in experimental allergic encephalomyelitis (EAE), a murine model of an autoimmune demyelinating disease. In this communication, we report that treatment of recipient mice with an antibody that neutralizes LT and TNF-alpha prevents transfer of clone-mediated EAE. LNC-8, a myelin basic protein-specific T cell line, produces high levels of LT and TNF-alpha after activation by concanavalin A, antibody to the CD-3 epsilon component of the T cell receptor, or myelin basic protein presented in the context of syngeneic spleen cells. LNC-8 cells transfer clinical signs of EAE. When LNC-8 recipient mice were also treated with TN3.19.12, a monoclonal antibody that neutralizes LT and TNF-alpha, the severity of the transferred EAE was reduced, while control antibodies did not alter the disease. The effect of anti-LT/TNF-alpha treatment was long lived and has been sustained for 5 mo. These findings suggest that LT and TNF-alpha and the T cells that produce them play an important role in EAE.

Animals↗
PubMed1990

The murine lymphotoxin gene promoter. Characterization and negative regulation.

Murine lymphotoxin (LT; TNF-beta) gene upstream regulatory elements were identified by linking fragments of 5' DNA to the chloramphenicol acetyl transferase gene. Fragment LT1 (-293 to +77 in relation to the proximal cap site) exhibited promoter activity which drove CAT expression in transfected murine fibroblasts and T lymphomas. Primer extension analysis of endogenous LT message confirmed that LT1 contained the necessary elements required for promoter function. Promoter activity was not observed when LT2 (-662 to +77), LT3 (-1186 to +77), or LT3 delta AX (-1186 to +77 (delta-662/-269)) were ligated to the chloramphenicol acetyl transferase gene and transfected into fibroblasts or T lymphomas. At least one upstream repressor element is postulated to account for this promoter inhibition. In contrast to the results obtained with fibroblast and T cell transfectants, LT1 was inactive in the B cell transfectants A20 and P3X63. This suggests that some B cells express a repressor factor that inhibits the LT promoter and/or they lack the necessary positive regulatory factors.

Animals↗
PubMed1990

Production of tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) by murine pre-B and B cell lymphomas.

Media from murine pre-B and B lymphoma cell cultures, but not from myeloma cell cultures, was cytotoxic to WEHI 164 cells, causing these TNF-sensitive targets to release 51Cr. The cytotoxic activity in the culture medium reached maximum levels approximately 4 days after the cell culture was initiated. The constitutive production of the factors was not influenced by depletion of serum from the medium or by the addition of either phorbol ester or bacterial endotoxin. The factor has a Mr greater than 10 kDa, and its cytotoxicity was abolished by anti-serum against murine TNF. Northern blot analysis with the use of cDNA probes to murine tumor necrosis factor (TNF-alpha) and lymphotoxin (LT, TNF-beta) showed high levels of TNF-mRNA in the pre-B cell lines, lower levels in the mature B cell lines and no TNF-mRNA in the myeloma cell lines. LT mRNA was present in pre-B cell lines, at a much lower concentration in only one of the B cell lines, and was not present in three other B lymphomas or in the myelomas tested. The results show a positive correlation between the presence of TNF and/or LT mRNA and the 51Cr-releasing activity present in the cell culture medium. Our data indicate that TNF and LT can be produced by murine B cells and that the synthesis of these cytokines may be restricted to certain differentiation stages of the B cell lineage.

Animals↗
PubMed1990

Expression of two distinct cytolytic mechanisms among murine CD4 subsets.

A TNF (TNF-alpha and TNF-beta)-sensitive target, L929, and two TNF-resistant targets, P815 and LK were used to compare the cytolytic activity among subsets of CD4+ (Th) clones. Cytolytic activity was induced with either Con A, CD3-mAb, or Ag-pulsed LK cells. Six Th1 clones are strongly cytolytic against all three targets. In contrast, Th2 clones are either noncytolytic or weakly cytolytic. Although there is an apparent correlation between TNF production, killing of L929 cells, and the killing of TNF-resistant targets, an anti-TNF serum (capable of neutralizing both TNF-alpha and TNF-beta) selectively inhibits CD4 clones to lyse L929 cells, whereas the lysis of P815 or LK cells was unaffected. The continuous presence of noncytotoxic levels of Actinomycin D (AcD) and cycloheximide, but not mitomycin C, cyclosporin A (CsA), or cholera toxin (ChT) inhibits the lysis of Ag-pulsed, Ia-bearing LK cells; indicating a requirement for de novo synthesis of RNA and protein for cytolytic activity. Although pretreatment with AcD, CsA, or ChT strongly inhibits production of IL-2, TNF and IFN-gamma, only clones pretreated with AcD lose cytolytic activity against Ag-pulsed, Ia-bearing LK cells. These observations support a model of TNF-independent killing of TNF-resistant targets. The TNF-independent cytolytic activity does not correlate with serine esterase activity released into media upon activation of CD4 clones. Moreover, the effects of metabolic inhibitors on serine esterase release do not correlate with their effects on cytolytic activity. Collectively, the data demonstrate that activated CD4 cells express two distinct cytolytic activities; a TNF (and IFN-gamma)-mediated cytotoxicity and a TNF (and IFN-gamma)-independent cytolytic activity. Both pathways require de novo synthesis of RNA and protein and appear to be independent of granule enzyme release. Only the TNF-independent cytolytic activity is resistant to CsA and ChT inhibition.

Animals↗
PubMed1990

Lymphotoxin and tumor necrosis factor-alpha production by myelin basic protein-specific T cell clones correlates with encephalitogenicity.

Lymphokine activity in seven myelin basic protein (MBP)-specific T cell clones was examined. All of the clones recognize MBP peptide 1-9 in the context of I-Au. A strong positive correlation was found between levels of lymphotoxin (LT) and tumor necrosis factor alpha (TNF-alpha) mRNA and biological activity on L929 cells and their capacity to induce paralysis, the clinical hallmark of experimental allergic encephalomyelitis (EAE). No correlation was found between interleukin-2 or gamma interferon production and encephalitogenicity. LT and/or TNF-alpha may play a central role in the pathogenesis of EAE.

Animals↗
PubMed1990

Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B.

Human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines constitutively produce high levels of biologically active lymphotoxin (LT; tumor necrosis factor-beta) protein and LT mRNA. To understand the regulation of LT transcription by HTLV-I, we analyzed the ability of a series of deletions of the LT promoter to drive the chloramphenicol acetyltransferase (CAT) reporter gene in HTLV-I-positive MT-2 cells. The smallest LT promoter fragment (-140 to +77) that was able to drive CAT activity contained a site that was similar to the immunoglobulin kappa-chain NF-kappa B-binding site. Since the HTLV-I tax gene activates the nuclear form of NF-kappa B, this finding suggested a possible means of HTLV-I activation of LT production. We found that the LT kappa B-like site specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2, C81-66-45, and other activated T cells. Mutation of the LT kappa B site in the context of the LT promoter (-293 to +77) (mutant M1) reduced the ability of the promoter to drive the CAT gene in HTLV-I-infected and noninfected human T-cell lines. These data suggest a general role for NF-kappa B activation in the induction of LT gene transcription. Activation of LT in HTLV-I-infected cells may explain the pathology associated with HTLV-I infection, including the hypercalcemia that is prevalent in adult T-cell leukemia.

Base Sequence↗
PubMed1989

Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor necrosis factors.

mAb to murine TNF (MuTNF) were produced after immunization of Armenian hamsters with purified, Escherichia coli-derived rMuTNF-alpha. Antibody produced from clone TN3-19.12, was purified and was found to inhibit 100% of the lytic activity of either recombinant or natural MuTNF-alpha at an antibody input of 25 ng/U. TN3-19.12 also inhibited all the lytic activity in culture supernatants from a variety of T cell sources, including activated T cell clones and T cell hybridomas (all of which expressed high levels of TNF-alpha and TNF-beta (lymphotoxin, LT) mRNA). Western blot analysis was used to document the physical form(s) of MuTNF recognized by TN3-19.12. Recombinant and macrophage-derived TNF displayed identical patterns of a single band with Mr 17 kDa. In contrast, T cell culture supernatants exhibited patterns consisting of two bands with Mr 17 and 24.7 kDa. The higher m.w. form was glycosylated based on its sensitivity to n-glycanase and displayed a m.w. consistent with that of TNF-beta (LT). These data suggest that TN3-19.12 recognizes both MuTNF-alpha and MuTNF-beta (LT). Monoclonal TN3-19.12 and polyvalent rabbit anti-rTNF were used to establish a MuTNF-specific ELISA capable of detecting picogram quantities of recombinant or natural TNF. This assay was used to detect TNF in the sera of mice challenged with a lethal dose of LPS. Peak TNF serum levels of 11 ng/ml were observed in these animals 90 min after i.p. LPS administration and then rapidly declined to near base line levels by 3 h. These values were confirmed by quantitating levels of TNF functional activity in the same samples. TN3-19.12 injected into mice subsequently treated with LPS prevented the detection of TNF in the circulation by either assay and protected mice from the lethal effects of endotoxin shock. Thus, TN3-19.12 effectively neutralizes endogenously produced TNF in vivo.

Animals↗
PubMed1988

Subtypes of helper cells. Non-inflammatory type 1 helper T cells.

Class II MHC-restricted T cells recently have been characterized as being either type 1 (Th1) or type 2 (Th2) based on their ability to both secrete different lymphokines and perform different functions. Characterization of these subtypes to date have indicated that Th1 cells secrete IL-2, IFN-gamma, lymphotoxin, and IL-3, whereas Th2 cells secrete IL-4, IL-5, and IL-3. Functionally, Th1 cells mediated cytotoxicity and delayed-type hypersensitivity, and have been termed "inflammatory cells," whereas Th2 cells mediate helper function for Ig secretion and have been termed, "regulatory cells." We now present evidence that not all Th1 clones are inflammatory and capable of mediating cutaneous delayed-type hypersensitivity. We have generated a number of myelin basic protein-specific Th1 clones that do not mediate swelling when injected together with myelin basic protein directly into the footpads of syngeneic mice. These results suggest that Th1 cells can be further subdivided based on their ability to mediate delayed-type hypersensitivity, and that the Th1/Th2 characterization of Th cells may be insufficient to adequately characterize all functional subtypes of class II MHC-restricted T cells.

Animals↗
PubMed1988

Lymphotoxin.

LT was one of the first lymphokines to be described and has been one of the most difficult to fit into a conceptual framework. Now, 20 years after its discovery, its structure, genetic organization, and linkage are well understood in mouse and human, and insight has been gained into its biological role. It is a T cell-derived glycoprotein of 25 kd coded by a gene within the MHC. It is somewhat (35%) structurally homologous to the macrophage product TNF. The genes for LT and TNF are tightly linked, and the proteins share most biological activities and compete for the same cell surface receptor. LT is induced in an antigen-specific MHC restricted fashion from class I and class II restricted T cells. Viral infection is also associated with LT production by lymphoid cells. LT has several effects on target cells including killing, growth stimulation, and induction of differentiation. The mechanism of LT's effects involves receptor binding and internalization and several sequelae including changes in prostaglandins and chromosome integrity. LT probably plays several biological roles. It can contribute to immunoregulation, defense against viruses, parasitic infections, and rejection of tumors. Understanding LT's role in the pathogenesis of diseases of autoimmunity and immune dysregulation will be the key to devising effective regimens for prophylaxis and treatment.

Amino Acid Sequence↗
PubMed1987

Cloning and expression of murine lymphotoxin cDNA.

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.

Amino Acid Sequence↗
PubMed1987

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. I. Destruction of B16 melanoma cells mediated by bystander cytolysis in vitro.

Two Lyt-1+, L3T4a+ autoreactive T cell clones specific for self-class II major histocompatibility complex (MHC) gene products were established from lymph node cells and spleen cells of C57BL/6J mice, respectively, by different methods. They were stimulated to proliferate in culture in response to I-Ab antigen-bearing syngeneic spleen cells in a class II MHC-restricted manner. This stimulation was inhibited completely by the addition of anti-L3T4a (GK1.5) or anti-I-Ab (3JP) monoclonal antibodies. The autoreactive T cell clones lysed syngeneic I-Ab+ target cells such as lipopolysaccharide (LPS) blasts. They also lysed I-A- bystander cells such as Cloudman and B16 melanoma and lymphoid tumor cells in the presence of I-Ab+ stimulator cells but not I-Ad+ cells. This bystander killing was most likely mediated by soluble factors released from the autoreactive T cells in response to I-Ab antigens, because culture supernatants from activated autoreactive T cells inhibited the proliferation of B16 melanoma cells in vitro and also had significant cytolytic activity. Both lymphotoxin and interferon-gamma were released from activated autoreactive T cells, suggesting that these cytotoxic lymphokines were responsible for autoreactive T cell-mediated cytolysis. The finding that the two clones, established independently and by different methods, show self-class II MHC antigen-restricted cytolysis, and bystander cytolysis suggests that these properties are not restricted to a unique population of autoreactive T cells. These results favor the concept that in vivo, autoreactive T cells may express not only regulatory activity in regard to antibody responses, but also anti-tumor activity via bystander cytolysis.

Animals↗