Tumor-specific suppressor cells induced by immunization with spleen cells from tumor-bearing animals.

Spleen cells from Fischer rats immunized with syngeneic spleen cells immune to the syngeneic 13762A mammary adenocarcinoma inhibited in vitro generation of lymphocytes cytolytic to the tumor. Spleen cells from rats immunized with nonimmune spleen cells were not suppressive. The suppressive property was first detected 10 days after immunization, persisted through the 17th day, and generally correlated with the appearance of an immunoglobulin G (IgG) factor blocking cell-mediated cytotoxicity to the tumor. Suppression was mediated chiefly by T-lymphocytes, but IgG-bearing lymphocytes also had some suppressive ability. Suppression was induced by IgG-positive cells or by serum or IgG from rats immunized to the 13762A mammary adenocarcinoma. The suppressor cells in the spleens of serum-immunized rats appeared earlier (3 days) than after immunization with immune spleen cells (10 days). These results suggest that certain IgG-positive spleen cells, as well as IgG present in the same tumor-bearing animals, induce one type of suppressor cell modulating cytolytic lymphocyte activity to this mammary adenocarcinoma.

Inhibition of DNA synthesis in cultured lymphocytes and tumor cells by extracts of betel nut, tobacco, and miang leaf, plant substances associated with cancer of the ororespiratory epithelium.

The high incidence of oropharyngeal, esophageal, and laryngeal cancers in certain parts of the world has been ascribed to conjugated tannins found in certain folk medicinal herbs. We extracted miang leaf and betel nut with phosphate-buffered saline (0.14 M NaCl, 0.15 M potassium phosphate buffer, pH 7.4) and found that the extracts inhibited [3H]thymidine incorporation by phytohemagglutinin-stimulated human lymphocytes and by rat mammary tumor and mouse L-cells in logarithmic growth. Pretreating the lymphocytes for 1 or 4 hr with the extracts inhibited phytohemagglutinin-induced thymidine incorporation 72 hr later. At concentrations of 2.5 volumes % or lower, miang and betel nut extracts inhibited thymidine incorporation by 40 to 98% without any apparent signs of toxicity as demonstrated by the 66Rb equilibrium assay. In addition, neither extract inhibited cytotoxicity of rat mammary tumor cells by immune syngeneic spleen cells. The molecular weights of the inhibitory factors were between 1,000 and 10,000 daltons as determined by ultrafiltration and were unaffected by boiling for 3 min or by treatment with alcohol and, therefore, are probably not proteins. This in vitro demonstration of inhibition of DNA synthesis by these plant extracts presumably enriched for conjugated tannins may relate to inhibition of growth of rats and chicks fed conjugated tanin-contaminated sorghum feed. The carcinogenic potential of either these extracts or conjugated tannins is not yet established.

T helper-cell phenotype regulates atherosclerosis in mice under conditions of mild hypercholesterolemia.

BACKGROUND: T cells are implicated in atherosclerosis, but little is known about the genetic control or molecular pathways, especially under conditions of mild hypercholesterolemia. METHODS AND RESULTS: BALB/c mice, making a CD4+ Th2 (IL-4+) cell response, express both MHC class II antigens (IA(d), IE(d)) and are atherosclerosis-resistant. C57Bl/6 mice produce a CD4+ Th1 (interferon [IFN]gamma+) response, express IA(b) but no IE, and are atherosclerosis-prone. To evaluate T helper-cell phenotype in fatty streak formation, wild-type C57Bl/6 mice (IA(b)+IE-) and transgenic mice, either AB(o), IA(b)-IE-; ABEalpha, IA-IE(k)+; or BL:TG:Ealpha, IA(b)+IE(k)+, were fed a high-cholesterol diet for 16 weeks and evaluated histomorphometrically for aortic lesions. Lesion size in AB(o), ABEalpha, and BL:TG:Ealpha strains was decreased by 54%, 79%, and 82%, respectively, compared with wild-type, correlating with decreased Th1 and increased Th2 expression and suggesting that T helper-cell phenotype is important in fatty lesion development. Decreasing Th1 cells by antibodies (alpha-CD4) or cytokines (IL-4) also caused >/=80% reductions in lesion size. Immunohistology revealed IFN-gamma, but not IL-4, colocalized with activated macrophages. Confirming these findings in a different mouse strain, BALB/c Stat 6 knockout mice (Th2 cell-deficient) developed aortic lesions comparable to C57Bl/6 mice on the same diet. CONCLUSIONS: In mildly hypercholesterolemic C57Bl/6 mice, presence of IA(b) and absence of IE regulated CD4+ T helper-cell phenotype; fatty lesions were proportional to IFNgamma+ Th1 cells in both C57Bl/6 and BALB/c strains. IFN-gamma may participate through macrophage activation, whereas IL-4 may act to limit Th1-cell response.

Helper-inducer T-lymphocytes mediate diabetes in EMC-infected BALB/c ByJ mice.

Diabetes mellitus developing in BALB/c ByJ mice infected with the M variant of encephalomyocarditis (EMC) virus is dependent on thymic immune mechanisms. We treated mice with the anti-T-lymphocyte monoclonal anti-L3T4 and anti-Lyt2.2 antibodies before virus inoculation and monitored the infection and occurrence of diabetes thereafter. Mice depleted of L3T4+ cells exhibited a reduced incidence and severity of diabetes compared with both untreated and anti-Lyt2.2-treated animals. All mice sustained pancreatic infections, but islet lesions with beta-cell degranulation only occurred in control infected and anti-Lyt2.2-treated animals. These data support the conclusion that the induction of diabetes in EMC-infected BALB/c mice is immune mediated and controlled by the L3T4 T-lymphocyte subpopulation.

Genetic influences on the immunologic pathogenesis of encephalomyocarditis (EMC) virus-induced diabetes mellitus.

DBA/2 and Balb/cBY mice were infected with approximately 30 plaque-forming units of the M-variant of encephalomyocarditis (EMC-M) virus. Seven days after inoculation the majority of the animals of both strains were hyperglycemic. A significant correlation between increased concentrations of virus in the pancreas and hyperglycemia was found among individual DBA/2 animals, but not among Balb/cBY mice. T-lymphocyte depletion of DBA/2 mice before infection failed to alter the incidence or severity of hyperglycemia in comparison to intact animals. Conversely, hyperglycemia in T-lymphocyte-depleted Balb/cBY mice was reduced substantially in comparison to infected immunocompetent animals. There appears to be at least two genetically influenced pathogenic mechanisms of diabetes in EMC-M virus-infected mice. In some strains of animals, hyperglycemia results exclusively from viral infection and the consequent injury to the beta cells, whereas in other animals, viral damage to the islets is compounded by immunologic events.

Dual functions of murine gammadelta cells in inflammation and autoimmunity in coxsackievirus B3-induced myocarditis: role of Vgamma1+ and Vgamma4+ cells.

Coxsackieviruses are a cause of clinical myocarditis. Both virus replication and host defense mechanisms, including virus-induced autoimmunity, mediate heart injury and cardiac dysfunction. Vgamma4+ cells kill infected cardiocytes and virus-specific CD4+ Th2 cells through Fas-dependent apoptosis and CD1d. The CD4+ Th1 response is necessary for activation of the autoimmune CD8+ T cells, which kill uninfected cardiocytes through perforin-dependent mechanisms.

T cells expressing the gamma delta T cell receptor induce apoptosis in cardiac myocytes.

OBJECTIVE: Enterovirus infections are major etiological factors in myocarditis and dilated cardiomyopathy. Using an experimental murine model of this disease, previous studies have shown that myocarditis susceptibility depends upon activation of T lymphocytes expressing the gamma delta T cell receptor (TcR), and that only mouse strains which accumulate gamma delta T cells in the myocardium show apoptosis of myocytes or evidence of dilated cardiomyopathy-like disease. The objective of the present studies is to demonstrate that gamma delta T cells directly induce greater Fas-dependent apoptosis of cultured myocytes than T cells expressing the alpha beta TcR. METHODS: Bl.Tg.E alpha mice were infected for 7 days with coxsackievirus B3 (CVB3). Hearts were removed and were either formalin-fixed, sectioned and stained with hematoxylin and eosin for inflammation, and using TdT-TUNEL for apoptosis, or were minced and collagenase digested for isolation of gamma delta+ and alpha beta+ T cells using immunomagnetic bead separation. Neonatal cultures of cardiac myocytes were isolated from mice less than 2 days old by collagenase and pancreatin digestion, and were either untreated or infected with virus. Levels of Fas (CD95) were measured using FITC-conjugated hamster anti-mouse Fas monoclonal antibody and flow cytometry. Susceptibility of myocytes to Fas-dependent killing was measured by 51Cr-release by labeled myocytes incubated for 4 h on either 3T3-mock or 3T3-FasL transfected cell monolayers. Killing by T cells was also measured in a 4 h 51Cr-release assay. Fas-dependent and perforin-dependent cytotoxicity was determined by specific blocking using either Fas-Fc or concanamycin A. RESULTS: Virally infected myocyte cultures showed significantly enhanced Fas expression compared to uninfected cells, with maximal upregulation of Fas occurring 18-24 h after virus infection. Both infected and uninfected myocytes were selectively killed by FasL-transfected 3T3 cells but not by mock control cells. Approximately 38% of CD3+ lymphocytes isolated from the heart express the gamma delta TcR with the remainder expressing the alpha beta TcR. Both gamma delta+ and alpha beta+ T cells lysed myocyte targets. Blocking studies indicate that gamma delta+ T cells induced predominantly Fas-mediated killing, while alpha beta+ cell produced more perforin-mediated death, although these effectors were capable of Fas-dependent killing as well. CONCLUSIONS: These studies demonstrate that T cells expressing the gamma delta TcR are more effective mediators of myocyte apoptosis than alpha beta+ T cells in vitro and suggests that these effectors may be primarily responsible for myocardial injury associated with dilated cardiomyopathy-like signs during coxsackievirus B3-induced myocarditis.

Apoptosis in coxsackievirus B3-induced myocarditis and dilated cardiomyopathy.

Group B coxsackieviruses (CVB), which infect the myocardium, cause myocarditis and dilated cardiomyopathy. However, not all infections of the myocardium result in disease. In the mouse model, CVB infection stimulates autoimmune T cell response to cardiac antigens, and these autoimmune effectors cause myocyte necrosis and cardiomyopathy. Induction of pathogenic autoimmunity depends upon CD4+ Th1 (interferon-gamma positive) cells while Th2 (IL-4 positive) cell responses promote disease resistance. T lymphocytes expressing the gamma-delta T cell receptor (gamma delta +) constitute up to 12% of the inflammatory cells in the heart and are crucial to maintaining a dominant Th1 response phenotype. gamma delta + lymphocytes modulate T cell responses by selectively lysing CD4+ Th2 cells. Th1 cells are not killed by gamma delta + cells. Lysis requires direct cell:cell interaction between the gamma delta + cell and CD4+ Th2 target and is most likely mediated through Fas:FasL interaction. These studies demonstrate a novel mechanism for immune modulation of cytokine responses in vivo.

Does the oxidative/glycolytic ratio determine proliferation or death in immune recognition?

Here we discuss the possibility that the way cells utilize fuel(s) for energy confers the properties that can be recognized by the immune system and, reciprocally, that recognition by the immune system can alter the balance of the cell's energy metabolism. We propose that immune recognition, of somatic cells via MHC can alter the their energy metabolism and induce a metabolic shift. We demonstrate the reciprocal relationship that inducing a shift in metabolism toward glycolysis by supplying glucose and insulin results in the upregulation of immunologically recognizable molecules such as cell surface Fas. Thus, immune recognition can induce metabolic deviation. Metabolic deviation can result in altered immune recognition and ultimately in cell proliferation, cell differentiation, or cell death.

The vascular endothelial cell is central to xenogeneic immune reactivity.

With the demand for organ transplants increasing, xenograft transplantation is being considered. A major obstacle to a solution to fulfilling this demand is xenograft rejection. Histologic evidence indicates that the vascular endothelial cell (VEC) is involved in both humoral and cellular aspects of xenograft rejection. To study this phenomenon murine VEC and splenocytes were used in a mixed lymphocyte/endothelial cell culture and in a mixed lymphocyte culture with human peripheral blood mononuclear cells as responders. The VEC is a much better stimulator than the splenocyte. Removal of macrophages and B cells from the human peripheral blood mononuclear cells has no effect on the response to the VEC. The VEC acts as a target for humoral responses in the complement-dependent cytotoxicity and the antibody-dependent cell-mediated cytotoxicity. The VEC is killed in these two assays, which indicates the importance of the VEC in humoral rejection. These data indicate that the VEC is an important cell in xenogeneic immune reaction and may be pivotal in xenograft rejection.

Cellular immune mechanisms in Coxsackievirus group B, type 3 induced myocarditis in Balb/C mice.

Coxsackie B viruses are a common cause of viral myocarditis in humans. A murine model of the human disease has been developed using Coxsackievirus group B, type 3 and inbred Balb/c mice. Infection of T lymphocyte deficient mice does not result in significant myocarditis indicating the importance of T cells in this disease. The virus can be isolated from the hearts of T cell deficient and normal mice in equal concentrations. Virus elimination presumably is mediated by virus specific neutralizing antibody induced in both groups. T lymphocytes, natural killer cells and macrophage obtained from normal virus infected mice are all capable of lysing myofibers in vitro. Maximum lysis is obtained with the cytolytic T cells. When these cell populations or Coxsackievirus immune antibody were adoptively transferred into T lymphocyte deficient animals infected with the virus, only animals given T cells developed significant myocarditis.

Cattle grazing a riparian mountain meadow: effects of low and moderate stocking density on nutrition, behavior, diet selection, and plant growth response.

Twelve ruminally cannulated and six intact crossbred beef steers were used in a randomized complete block design to evaluate the effects of stocking density of a riparian pasture in the Sierra Nevada mountains on grazing behavior, dietary selection, forage intake, digesta kinetics, and growth rates of Carex nebraskensis and Juncus balticus. Nine .5-ha pastures were assigned to one of three treatments: ungrazed (CON) or grazed to leave either 1,500 kg/ha (LOW) or 1,000 kg/ha (MOD). Two collections were conducted during the summer of 1992 (following winter drought) and 1993 (following above-average winter precipitation). Standing crop biomass was greater (P < .05) in grazed pastures than in CON pastures at initiation of grazing in 1992 but not in 1993. After grazing in both 1992 and 1993, a treatment x intrapasture location interaction was noted (P < .05). Tiller growth rates in both 1992 and 1993 were affected (P < .05) by a treatment x growth period interaction. Stocking density did not alter (P > .10) botanical or chemical composition of the diet in 1992, and only minor differences were noted (P < .05) in 1993. Forage intake, passage rate measures, and total time spent loafing did not differ (P > .10) between LOW and MOD steers. Within the mid-meadow area in 1992, loafing time was greater (P< .05) for MOD steers than for LOW steers. In 1993, a treatment x trial interaction was noted for loafing time in all three areas. Total time spent grazing was greater (P < .05) for MOD steers than for LOW steers in 1992 and was affected (P < .05) by a treatment x trial interaction in 1993. In 1992 grazing time along the streamside was greater (P < .05) for LOW steers than for MOD steers, and significant treatment x trial interactions were noted for grazing time spent along the forest edge and mid-meadow areas. In 1993, only streamside grazing time was influenced by treatment being greater (P < .05) for MOD steers than for LOW steers. In general, our data suggest that management decisions to reduce stocking densities may force cattle to congregate along streambanks and to concentrate grazing and loafing activities in those areas.

Supplemental cracked corn or wheat bran for steers grazing endophyte-free fescue pasture: effects on live weight gain, nutrient quality, forage intake, particulate and fluid kinetics, ruminal fermentation, and digestion.

Two experiments were conducted with beef steers (Exp. 1, average BW of 580 kg; Exp. 2, average BW of 247 kg) to evaluate the use of no supplements (CON) or daily supplementation with (OM basis) .34% of BW of cracked corn (CORN), .34% of BW of wheat bran (WBBW), or .48% of BW of wheat bran (WBISO; calculated to be isocaloric to CORN) on digestive responses (Exp. 1) and live weight gain (Exp. 2). In Exp. 1, type of supplement did not affect (P > .10) the dietary fiber or N constituents, but in vitro OM disappearance of the forage differed (P < .10) with supplementation and type of supplement fed. Supplemented steers consumed less (P < .05) forage and total OM. Particulate passage, fluid passage, and ruminal pH were not affected (P > .10) by supplementation. Ruminal NH3 N concentration showed (P < .05) a treatment x sampling time interaction and, in general, WBBW and WBISO steers had greater ruminal NH3 N than CORN and CON steers. Total VFA concentrations and molar proportions of propionate were lower (P < .10) in CON steers than in supplemented steers; no differences were noted (P > .10) among supplemented steers. Molar proportions of acetate were lower (P = .01) in supplemented steers than in CON steers and were greater (P = .03) in WBBW steers than in WBISO steers. Butyrate molar proportions were lower (P < .05) in CON steers than in supplemented steers and differed (P < .10) with type and quantity of supplement supplied. In situ forage NDF disappearance at 6, 9, and 24 h after feeding and rate of disappearance were greater (P < .05) in CON steers than in supplemented steers. In Exp. 2, CON steers weighed less (P = .01) than supplemented steers, CORN steers weighed more (P = .08) than wheat bran-supplemented steers, and WBISO steers weighed more (P = .02) than WBBW steers; ADG for 90 d followed a similar response. Results suggest that supplementation of wheat bran rather than corn did not seem to stop the reduction in forage intake or OM digestion associated with corn supplementation.

Pretanned leather shavings in a supplement mixture for steers: I. In situ and in vitro disappearance, ruminal fermentation, and organic matter, nitrogen, and fiber digestion.

Two digestion studies were conducted to evaluate the use of pretanned leather shavings as a component of a protein supplement. In Exp. 1, the in situ and in vitro disappearance of pretanned leather shavings and soybean meal was evaluated. Results revealed that less than 18.4% of the pretanned leather shavings was solubilized and disappeared when exposed to McDougall's buffer for 48 h, but there was 90.0% disappearance with 48-h exposure to a .1 N HCl/pepsin treatment and 97.0% disappearance with exposure to a two-stage digestion. In situ disappearance following 72 h in the rumen allowed 6.8% disappearance. Thus, leather shavings seem to be relatively indigestible in the rumen, but postruminal digestion may be possible. In Exp. 2, six Angus x Holstein steers, fitted with ruminal and duodenal cannulas, were used in a replicated 3 x 3 Latin square to evaluate ruminal and digestion effects of the following supplements combined with fescue hay at 1.7% of BW (DM basis): no supplementation (control); supplementation intraruminally with soybean meal at .07% of BW (as-fed basis); and supplementation intraruminally with a combination of soybean meal and pretanned leather shavings (17:8 ratio) at .05% of BW (isonitrogenous to soybean meal; as-fed basis). Ruminal fluid passage rate was greater and fluid turnover time was shorter in steers fed leather shavings than in those fed soybean meal (P = .10). Ruminal pH was lower (P = .04) for supplemented steers than for control steers and ruminal NH3 N concentration was greater (P = .01) in steers fed soybean meal than in those fed leather shavings. Total VFA concentration was increased (P = .02) by supplementation. Supplementation with soybean meal increased (P < .05) ruminal molar proportions of butyrate, valerate, and isovalerate compared with leather shavings. Duodenal OM flow and OM disappearing in the intestines were increased by supplementation (P < .10), but not by the type of supplement fed (P > .10). Ruminal digestion of OM and total tract OM digestion were unaffected (P > .10) by supplementation and the type of supplement fed. Flow and digestion of NDF were unaffected (P > .10) by the treatments. Flow of N and the quantity of N disappearing in the intestines were increased (P < .05) by supplementation but did not differ (P > .10) between supplementation groups. Microbial N flow, N utilization for net microbial protein synthesis, and ruminal N disappearance were unaffected (P > .10) by supplementation and the type of supplement provided. Combining pretanned leather shavings with soybean meal seemed to have no deleterious effects on digestion or fermentation and to allow for escape of some N to the lower tract.