Detection of the MAIDS virus using the polymerase chain reaction.
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3T3 Cells↗
Biomedical subjects
R A Good
Publications and source records attributed to R A Good.
Influence of a small number of mature T cells in donor bone marrow inocula on reconstitution of lymphoid tissues and negative selection of a T cell repertoire in the recipient.
Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1a) reactive V beta 6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T- chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T+ chimeras). When lethally irradiated AKR (Mls-1a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T+ and T- chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T+ chimeras but not in T- chimeras. On the other hand, clonal elimination of V beta 6+ T cells reactive to the recipient Ag (Mls-1a) was abolished in T+ chimeras but successfully induced in T- chimeras. The V beta 6+ T cells not eliminated in T+ chimeras showed depressed responses against Mls-1a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.
Animals↗
Suppression of T-cell proliferation by CD8+ T cells induced in the presence of protoscolices of Echinococcus multilocularis in vitro.
Immunoregulatory influences of protoscolices (PSC) of Echinococcus multilocularis on murine T-lymphocyte functions have been examined in an in vitro system. Proliferative responses of spleen cells stimulated with concanavalin A (ConA) or anti-CD3 monoclonal antibodies were depressed by the addition of PSC. In the presence of PSC, both interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression by lymphocytes stimulated with ConA were significantly reduced. However, exogenous IL-2 reconstituted both the ConA-stimulated proliferative responses and IL-2R expression. These findings suggest that PSC of E. multilocularis can suppress lymphoid cell responses via influences on IL-2 production. Indeed, addition of CD(8+)-enriched cells from cultures stimulated with ConA plus PSC to fresh spleen cells showed marked suppression of the ConA responses. IL-2 production as well as IL-2R expression on the spleen cells so treated were suppressed. These findings reveal a suppressive immunologic function induced by E. multilocularis PSC that involves inhibition of IL-2 production and reduction of IL-2R expression. The PSC-induced CD8+ cells appear to play a key role in the suppressive regulation of host immune responses against E. multilocularis.
Animals↗
Preventive action of carotenoids on the development of lymphadenopathy and proteinuria in MRL-lpr/lpr mice.
The chemopreventive action of carotenoids on proteinuria and lymphadenopathy were examined in autoimmune-prone MRL-lpr/lpr (MRL/l) mice. They were fed a synthetic full-fed diet (16-18 kcal/mouse/day) with supplementation of beta-carotene or astaxanthin (0.19 mumoles/mouse, 3 times a week), and the development of lymphadenopathy and proteinuria were examined. MRL/l mice fed a full-fed diet without the supplementation of carotenoids or those fed a calorie-restricted (CR) diet (10-11 kcal/mouse/day, 60% calorie intake of full-fed mice) were employed as controls. CR dramatically delayed the development of proteinuria and lymphadenopathy, as reported previously. Carotenoids also significantly delayed the onset of these symptoms in MRL/l mice fed a full-fed diet. Carotenoids were half as effective as CR and astaxanthin, a carotenoid without provitamin A activity, which appeared to exert more significant preventive actions than beta-carotene in delaying the development of these symptoms. Similar chemopreventive actions of carotenoids were also demonstrated in MRL/l mice fed a regular diet (Lab Chow). CR has been shown to augment IL-2 production and to decrease serum prolactin levels in this strain, which may be related to its dramatic preventive action of autoimmunity. However, carotenoids did not affect IL-2 production nor prolactin levels in full-fed MRL/l mice. The chemopreventive actions of carotenoids observed in autoimmune-prone MRL/l mice may be attributed to yet unknown mechanisms, apart from their provitamin A activity or oxygen-quenching activity.
Animals↗
Calories, cell proliferation, and proviral expression in autoimmunity and cancer.
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Animals↗
Molecular cloning and characterization of a cDNA encoding feline interleukin-6.
With the discovery that the cat is susceptible to infection by a lentivirus quite similar to the virus that causes the human acquired immune deficiency syndrome, the cat has become an important animal model with which the interactions between host and immunodeficiency virus can be studied. To facilitate investigation of the feline immune response and the response of cytokine expression to infection by feline immunodeficiency virus, our laboratory has focused on the isolation and molecular cloning of cDNA representative of feline lymphokines, cytokines, and monokines. Herein, we describe the molecular cloning and characterization of a cDNA encoding feline interleukin (IL)-6. The nucleotide sequence of feline IL-6 was found to exhibit 81%, 76%, 63%, and 61% homology with pig, human, rat, and mouse IL-6, respectively, while the predicted amino acid sequence exhibits 66%, 53%, 37%, and 30% homology with pig, human, rat, and mouse IL-6, respectively.
Amino Acid Sequence↗
Generation of CD8+ suppressor T cells by protoscoleces of Echinococcus multilocularis in vitro.
Addition of protoscoleces (PSC) of Echinococcus multiocularis suppressed proliferative responses in spleen cells stimulated with concanavalin A (Con A) on day 3 of culture. The suppression was not observed in the spleen cell population depleted of CD8+ cells but observed in the population depleted of CD4+ cells, suggesting the involvement of the CD8+ T cells in the apparent suppression. Indeed, flow cytometry analysis revealed that the proportion of CD8+ cells markedly increased in the Con A cultures containing PSC. Furthermore, when spleen cells were co-cultured with PSC alone, marked increase in the proportion of CD8+ cells as well as B220+ cells was observed. Addition of these increasing CD8+ cells suppressed the proliferative responses of fresh spleen cells stimulated with Con A. These findings suggest that the low responsiveness of spleen cells to Con A on day 3 of culture in the presence of PSC is attributable to an active suppressor mechanism including CD8+ T-suppressor cells generated by stimulation with PSC.
Animals↗
Human prolactin regulates transfected MMTV LTR-directed gene expression in a human breast-carcinoma cell line through synergistic interaction with steroid hormones.
Prolactin plays a key role in the regulation and growth of mammary cells, and influences tumor promotion. We have shown that chronic energy restriction intake depresses prolactin levels, inhibits production of MMTV proviral DNA and proto-oncogene expression in mammary glands and prevents development of mammary tumors. Since the expression and proto-oncogene activation of MMTV are regulated by promoter/enhancer elements within its long terminal repeat (LTR), in the present study we used a chloramphenicol acetyl transferase (CAT) reporter gene system and gene transfection methods to study the effect of prolactin on MMTV LTR using a human ductal carcinoma cell line T47D stably or transiently transfected with a plasmid consisting of the LTR upstream of CAT gene. Human prolactin or dexamethasone induced, respectively, a 2-fold or 6-fold increase in CAT activity compared with background CAT activity in the absence of hormones. However, the combination of human prolactin and dexamethasone strongly enhanced (20-fold) induction of the LTR compared with the control. Human prolactin also showed a synergistic effect with progesterone on LTR induction. Both LTR and CAT genes needed to be linked for induction of CAT activity by prolactin and dexamethasone. Our results indicate that human prolactin can act synergistically with steroid hormones to regulate MMTV LTR-directed gene expression in transfected T47D cells.
Breast Neoplasms↗
A strategy for making synthetic peptide vaccines.
We have determined the H-2 class II allele-specific amino acid motif of the agretope (the site of contact between the peptide antigen and the major histocompatibility complex) for a synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58). Residues 46 and 54 functioned as the agretope, and residues 50 and 52 functioned as the epitope (the site for contact between the peptide antigen and the T-cell antigen receptor). In general, agretopes and epitopes function independently. Thus, substitution of amino acids in the epitope does not significantly affect binding of the peptide antigen to a class II molecule. On the basis of these findings, synthetic peptide vaccines against influenza Aichi (H3N2) virus were prepared by introducing seven residues of the influenza virus hemagglutinin into the frame component residues 43-46 and 54-58 of p43-58 analogues including the agretopes for Ak or Ab previously determined on the p43-58 segment. These peptide vaccines induced both helper T-cell responses and production of antibodies that were specific for influenza Aichi hemagglutinin but not for the major histocompatibility complex binding frame in mice bearing Ak or Ab. The antibodies produced neutralize the infectivity of influenza Aichi in vitro. The present findings should provide a basis for preparing potent peptide vaccines that function without producing side effects.
Alleles↗
Superantigen staphylococcal enterotoxin B-induced T-helper cell activation is independent of CD4 molecules and phosphatidylinositol hydrolysis.
The role of the CD4 molecule in activation of T-helper cells was examined by investigating the effect of an anti-CD4 monoclonal antibody (Leu3a) in conventional peptide antigen-specific cloned T-helper cells that are also reactive to staphylococcal enterotoxin B (SEB). These T-helper cell clones are CD4+/CD45RO+/T-cell antigen receptor beta-chain variable region 12-positive and can respond to nominal peptide antigens and SEB by proliferation in the presence of class II major histocompatibility complex-expressing accessory cells. Although antigen and SEB were comparable in their ability to induce proliferative responses, interleukin 2 (IL-2) production, and IL-2 receptor alpha-chain expression, stimulation with SEB failed to trigger phosphatidylinositol hydrolysis or a rise in the intracellular free calcium ion concentration. Leu3a treatment inhibited antigen-induced proliferative responses of T cells with concomitant suppression of IL-2 production and IL-2 receptor expression. In contrast, SEB-induced responses were unaffected by Leu3a. These findings indicate that the functional consequences of binding (ligation) of conventional antigen and of superantigen with the T-cell receptor are distinct in the context of both signal transduction pathways and participation of CD4 molecules.
Antibodies, Monoclonal↗
An NK1.1+ CD4+8- single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor V beta family.
In the present report we describe a CD4+8- heat stable antigen-negative (HSA-) thymocyte subpopulation that expresses a distinguishably low density of alpha beta T-cell antigen receptors (TCRlo) from the majority of CD4+8- high-density TCR (TCRhi) mature-type thymocytes. This subpopulation appears relatively late in life. Analysis of MEL-14, Pgp-1 (CD44), ICAM-1 (CD54), and NK1.1 expression on this subpopulation revealed that the CD4+8- TCRlo population was a population having unique characteristics (MEL-14-, CD44+, ICAM-1+, and NK1.1+) compared to the CD4+8- TCRhi thymocytes, most of which are MEL-14+, CD44-, ICAM-1-, and NK1.1-. When TCR beta-chain variable region (V beta) usage was analyzed, this thymic population expressed predominantly products of V beta 7 and V beta 8.2 TCR gene families. Interestingly, cells with V beta 8.1 TCRs, which are reactive to Mls-1a antigens, were not eliminated from the CD4+8- HSA- TCRlo subpopulation but had been eliminated from the major CD4+8- HSA- TCRhi subpopulation in Mls-1a strains. A subset with a phenotype similar to the CD4+8- HSA- TCRlo thymocytes was also identified primarily in bone marrow, and this subset constituted approximately half of the CD4+ T cells in the bone marrow. The CD4+8- HSA- TCRlo cells showed extremely high proliferative responses to immobilized anti-TCR antibody but generated negligible responses to allogeneic H-2 antigens compared to the responses generated by the major CD4+8- HSA- CD3hi cells. However, the CD4+8- HSA- TCRlo cells in Mls-1b mice mounted vigorous proliferative responses to Mls-1a antigens but not in Mls-1a mice. The properties of this T-cell subset suggest that these cells belong to a lineage distinct from the major T-cell population.
Age Factors↗
Improved enzyme immunosorbent assay for mouse prolactin using penicillinase as label.
Enzyme immunosorbent assay (EIA) for mouse prolactin was established by modifying a method originally developed for human prolactin by Shrivastav et al. This simple, sensitive, rapid, and reproducible assay utilizes penicillinase as the labeling enzyme, rabbit anti-mouse prolactin antibody (Ab) and goat anti-rabbit Ig Ab as the first and second antibodies. Prolactin reference preparations and enzyme-conjugated prolactin were mixed with the first Ab and incubated for 0.5 h at 4 degrees C (24-48 h for serum samples). Then, the sample mixture was transferred to the wells of microtiter plate coated with the second Ab. After being kept at room temperature for 2 h, the plate was washed and filled with substrate solution (penicillin V). Absorbance at 620 nm was measured with an ELISA reader to quantitate the amount of conjugated prolactin bound to the second Ab. The prolactin levels obtained by this assay exhibited good correlation with those measured by radioimmunoassay (RIA) (y = 0.95x + 9.14, r = 0.943), and the sensitivity of EIA was equivalent to that of RIA (1.7 ng/ml). The CVs of intra-assay and inter-assay by EIA for mouse serum samples ranged comparably to those by RIA.
Aminoquinolines↗
Influence of calorie restriction on oncogene expression and DNA synthesis during liver regeneration.
Controlling calorie intake (CCI) extends healthful life-span by a mechanism that may involve reduced rates of cell division without detriment to inducible cellular responses. To test whether inducible cellular proliferation is preserved by CCI and whether the mRNA expression levels of oncogenes activated by cell division can be reduced by CCI, we evaluated the effect of dietary energy on the hepatocellular proliferative burst and on oncogene and growth factor mRNA expression induced by partial hepatectomy. Eighty Fischer 344 rats were separated into two dietary groups and were fed semipurified diets for 10 weeks that differed only in calories by 40%. Mean hepatic levels of [3H]thymidine incorporation were greater among CCI animals at 18, 24, 28, and 36 hr after partial hepatectomy. The expression of c-fos and c-Ki-ras mRNAs, activated during hepatic regeneration, was reduced by CCI. Peak expression of c-fos among ad libitum fed controls to levels 4-6 times greater than prehepatectomy levels was not detected among CCI animals. Protracted elevated expression of c-Ki-ras among ad libitum fed animals was foreshortened by CCI. These findings demonstrate that inducible cellular proliferative responses are preserved by CCI and that the mRNA expression levels of c-fos and c-Ki-ras activated during cell division are reduced by controlling dietary energy. Preserved inducible cellular responses and lowered oncogene expression during cell division may be attributes of the healthful protective effect of CCI.
Animals↗
The effects of dietary restriction on immune function and development of autoimmune disease in BXSB mice.
Chronic energy intake restriction (CEIR) prolonged the median life span and inhibited autoimmunity and development of autoimmune disease in BXSB mice, as has been established for mice of several other autoimmune-prone, short-lived strains. Whether imposed just after weaning or delayed until manifestations of disease had appeared, CEIR inhibited or reversed development of autoimmunity and immune complex-based renal disease in male BXSB mice. CEIR also prevented the formation of anti-DNA antibodies and prevented the increase in circulating immune complex levels that is typically observed in male mice of this strain. Moreover, CEIR inhibited development of splenomegaly and prevented the normal age-associated decline of a number of immunological functions, including interleukin 2 production, cell-mediated cytotoxic responses, and mixed lymphocyte reactivity. The observed improvement in cell-mediated immune responses was attributed largely to the capacity of CEIR to inhibit development of the splenomegaly that occurs concomitant with expansion of a non-T, non-B lymphoid cell population. These findings emphasize that CEIR, even when imposed relatively late in life in BXSB mice, can influence expression of autoimmunities and autoimmune diseases of different genetic origins and presumed pathogenetic bases.
Age Factors↗
Induction of heat shock protein closely correlates with protection against Toxoplasma gondii infection.
Heat shock proteins (HSPs) are evolutionarily highly conserved polypeptides that appear to be produced by many cells to preserve cellular functions under a variety of conditions of stress, including infections. We report that a 65-kDa HSP is present in mouse peritoneal cells that have been infected with a low-virulence (Beverley) strain of Toxoplasma gondii, as determined by electroblot assay using a monoclonal antibody specific for microbial HSP65. This HSP is, however, not expressed when infection occurs with the high-virulence RH strain of T. gondii. Furthermore, HSP was demonstrable in mice that acquired resistance against infection with a lethal dose of bradyzoites of the Beverley strain or even of an inoculum of a highly virulent strain of T. gondii (RH). From these results, it can be suggested that HSPs play an important role in developing effective defenses that include effective immune responses against infection with Toxoplasma parasites in vivo.
Animals↗
A synthetic peptide homologous to retroviral envelope protein down-regulates TNF-alpha and IFN-gamma mRNA expression.
We investigated the influence of CKS-17, a synthetic heptadecapeptide that corresponds to a highly conserved domain of the immunosuppressive retroviral envelope protein p15E, on staphylococcal enterotoxin B (SEB)-induced TNF-alpha gene expression in human peripheral blood mononuclear cells and highly purified human monocyte preparations, as well as the production of TNF-alpha protein, using human peripheral blood mononuclear cells. RNA hybridization studies show that CKS-17 inhibits SEB-induced TNF-alpha mRNA accumulation in human peripheral blood mononuclear cells and human monocytes. CKS-17 is also shown to be highly suppressive for SEB-induced production of TNF-alpha proteins. Similarly, CKS-17 inhibits expression of SEB-induced IFN-gamma mRNA in human peripheral blood mononuclear cells. These results suggest that CKS-17 down-regulates both TNF-alpha and IFN-gamma production at mRNA level.
Amino Acid Sequence↗
Suppression of human interferon-gamma production by a 17 amino acid peptide homologous to the transmembrane envelope protein of retroviruses: evidence for a primary role played by monocytes.
CKS-17, a synthetic amino acid peptide homologous to a highly conserved region of retroviral transmembrane protein exerts a suppressive action on staphylococcal enterotoxin A (SEA)-induced the production of IFN-gamma by human peripheral blood mononuclear cells (PBMC) (Ogasawara et al., J. Immunol. 141, 615, 1988). This action has been shown in the present study to be preceded by dramatic clustering of PBMC. Clusters appear within 3 hr of exposure of PBMC to CKS-17; they are dose dependent, inhibited by cycloheximide, and require a temperature of 37 degrees C. The cells in the clusters are predominantly monocytes. Although it has been previously shown that CKS-17 inhibits monocyte-mediated killing by inactivating IL-1 (Kleinerman et al., J. Immunol. 139, 2329, 1987) and production of IL-2 by murine thymoma cells treated with IL-1 (Gottlieb et al., J. Immunol. 142, 4321, 1989), in the present study we show that IL-1 does not prevent clustering of PBMC by CKS-17. Using CKS-17 and highly purified monocytes or lymphocytes, profound alterations occur only with monocytes, as revealed by light or electron microscopy. SEA- or staphylococcal enterotoxin B-induced production of IFN-gamma is inhibited when highly purified monocytes pretreated with CKS-17 are cocultured with highly purified T lymphocytes. Thus, CKS-17 induces dramatic clustering of cells apparently by inducing alterations of monocytes but not lymphocytes, suggesting that CKS-17 may interfere with the capacity of monocytes to facilitate production of IFN-gamma by T lymphocytes.
Amino Acid Sequence↗
Effects of K-76COOH (MX-1) on immune response: induction of suppressor T-cells by MX-1.
K-76COOH (MX-1), isolated from the cultured supernatant of a species of fungi imperfecti, Stachybotrys complement nov. sp. K-76, is an inhibitor of the complement component, C5. The effects of MX-1 on various immune responses were investigated. MX-1 enhanced the response of spleen cells to PHA and LPS: MX-1 at 0.01-250 micrograms/ml for PHA and at 10-250 micrograms/ml for LPS. In contrast, it inhibited the response to Con A: MX-1 at 0.01-500 micrograms/ml for spleen cells and at 100-500 micrograms/ml for thymocytes. MX-1 and IL-1 synergistically acted to enhance the Con A response of spleen and thymus cells from which accessory cells and Ia-positive cells had been removed by passing through Sephadex G-10 columns and treating with anti-Ia monoclonal antibody plus complement. T-cells pretreated with MX-1, IL-1 and Con A for 3 days suppressed not only the response of B-cells to LPS but also the production of anti-SRBC antibodies. In addition, MX-1 was found to increase CD8+ T-cells. These results suggest that MX-1 acts on T-cells to induce suppressor T-cells.
Animals↗