Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.     

Effects of endotoxin on immunity in aging mice

In vivo administration of bacterial lipopolysaccharides (LPS) to young adult mice causes a dose-dependent depression of antibody formation. In contrast, LPS produced no effects on the responses of older mice and these animals appeared to be refractory to the depressive effects observed in the young adults. When young adults were pretreated with endotoxin their baseline control responses were also severely depressed and the LPS response profile resembled that of the normal 9-month-old animals. These results suggest that endotoxin effects may contribute to the genesis of immune abnormalities in the aged.     

Characterization of cytostatic effector lymphocytes during the development of a syngeneic lymphosarcoma in C3H mice: use of monoclonal reagents to identify T-cell subsets

The development of the in vitro cytostatic capacity of splenic lymphocyte subpopulations from C3H mice carrying the syngeneic Gardner tumor was examined at different times after intramuscular tumor injection. Most mice died between 3 to 6 weeks after tumor injection, while some rejected their tumors or survived longer than 3 months. Cell separation procedures and monoclonal antibodies against T-cell subsets were used to identify the cells responsible in anti-tumor immunity. Cytostatic capacity against tumor cells developed in the T-cell enriched subpopulation of splenocytes 3 days after tumor injection and was partly abrogated by anti-Lyt-1. Effector function of Lyt-2+ T cells and B cells developed later and peaked at around 10 days after tumor injection. Another cell population with cytostatic capacity which was not blocked by anti-Lyt-1, anti-Lyt-2, or anti-Ly-5 was noted to develop early after tumor injection and lacked both T-cell and B-cell markers ("null"). This subpopulation was eluted with T cells from nylon wool columns and comprised up to 50% of the T-enriched fraction of splenocytes in later stages of tumor growth. An interesting characteristic of these "null" cells was susceptibility to T-cell suppression both in early and later stages of tumor growth except in regressor mice which lacked suppressor T cells. The cytostatic capacity of the "null" cells could be restored either by removal of Thy-1+ cells from the T-enriched fraction by panning, or the addition of anti-Thy-1 or F(ab')2 fragments of anti-Thy-1 to the lymphocyte-tumor reaction mixtures. Most mice examined after 10 days of tumor growth were immunosuppressed to varying degrees. Unseparated splenocytes from these mice were not cytostatic but removal of T cells allowed the B cells to exert their cytostatic capacity. A strong underlying B-cell cytostasis was shown to be present in long survivor mice even though their unseparated spleen cells were only weakly cytostatic. T cells did not play a role in the regression of tumors or long-term survival of tumor bearer mice. Splenocytes from regressor mice were strongly cytostatic, their anti-tumor activity residing in the "null" and B-cell populations.     

T-cell subsets in the thyroids of mice developing autoimmune thyroiditis

To examine the role of T-cell subsets in the development of thyroid lesions, female CBA/J mice were immunized with 60 μg mouse thyroglobulin (MTg) in 0.1 ml complete Freund's adjuvant in both hind footpads. The thyroids were removed 12–21 days later, pooled, and dispersed. The cell suspension was examined by membrane immunofluorescence for the distribution of Thy-1⁺, Lyt-1⁺, Lyt-2⁺, and sIg⁺ lymphocytes. For comparison, peripheral blood leukocytes (PBL) from the same animals were similarly examined. Throughout this 10-day interval, B cells in the thyroid were consistently below 5%, whereas B cells represented 19–24% of PBL. Thy-1⁺ cells in PBL ranged from 45 to 59%, whereas Thy-1⁺ cells in the thyroid were 37–50%. However, only thyroidal T cells showed a consistent decline with time and were replaced gradually by cells without T or B cell markers. In particular, there was a clear shift in the Lyt-1⁺:Lyt-2⁺ ratio from about 7 down to 2 in the thyroid as the early predominance of Lyt-1⁺ cells was followed by a relative increase in Lyt-2⁺ cells. Our results show that there is an accumulation of Lyt-1⁺ and Lyt-2⁺ cells in the infiltrated thyroid. These cells may include MTg-reactive, helper, and cytotoxic T cells which localize (or differentiate) in the thyroid and initiate the lesions.     

Defective spontaneous but normal antibody-dependent cytotoxicity for an extracellular protozoan parasite, Giardia lamblia, by C3H/HeJ mouse macrophages

To understand murine host responses to extracellular protozoa, the capacity of peritoneal macrophages to exhibit cytotoxicity for [3H]thymidine-labeled Giardia lamblia trophozoites was investigated. Resident peritoneal macrophages from C3H/HeN mice expressed spontaneous cytotoxicity for G. lamblia in a manner that was dependent on both time and effector cell number; this cytotoxic activity was increased with cells elicited by an intraperitoneal injection of thio-glycollate. In contrast, spontaneous cytotoxicity for G. lamblia by resident and thioglycollate-elicited peritoneal macrophages from C3H/HeJ mice was markedly reduced. In the presence of anti-G. lamblia serum (ADCC), however, peritoneal macrophages from both C3H/HeN and C3H/HeJ mice exhibited striking augmentation of their cytotoxic activity for G. lamblia to equivalent levels. We conclude that macrophages from C3H/HeJ mice express defective spontaneous cytotoxicity but normal ADCC for the extracellular protozoan parasite, G. lamblia. The dissociation between the expression of these two effector cell functions suggests that macrophage spontaneous cytotoxicity and ADCC for extracellular protozoa are mediated by separate macrophage functions.     

In vitro inhibition of the helper activity of GAT-specific T-cell lines by a syngeneic anti-idiotypic serum: preferential effect on the IgG response

We described in this paper the characteristics of a syngeneic anti-idiotypic serum made in BALB/c against BALB/c anti-poly (Glu⁶⁰ Ala³⁰ Tyr¹⁰) (GAT) antibodies. This serum recognizes idiotypic determinants present in all anti-GAT sera whatever the allotypic markers of the mice used to prepare the sera. The functional effect of this serum on two helper cell lines is also described. Cell line BDF₁/52 was obtained from GAT immunized lymph node cells (LNC). Cell line BDF₁/E₃ was selected from splenic T-cells educated in vitro on GAT-pulsed adherent cells. Both lines were propagated in presence of filler cells, antigen, and medium containing T-cell growth factor(s) from splenic cells activated with concanavalin A. Both cell lines exhibit a helper activity as measured by the plaque-forming cell (PFC) response they induce in vitro in the presence of DNP-GAT and DNP sensitized B cells. Their helper activity is specific and they require a hapten-carrier bridge to activate B cells. These lines are able to induce IgG₁, IgG_2a and IgG_2b anti-TNP PFC. Syngeneic anti-idiotypic serum B 658 inhibits specifically the function of these two lines but does not affect the helper activity of an OVA-specific T-cell line. The blocking activity of the serum can be adsorbed on a hybridoma protein with anti-GAT activity. This inhibition affects more dramatically the IgG₁ response than the IgG_2a and IgG_2b responses.     

Affinity and distribution of subpopulations of antibody-producing cells in protein-restricted C57BL/6 mice

To study the effect of protein restriction on the affinity of antibodies produced by plaque-forming cells (PFC), C57BL/6 mice were fed diets containing 4% (R4%), 8% (R8%), or 27% (N) casein for 2 (short-term) or 12 (long-term) weeks and immunized with dinitrophenyl (DNP) bovine gamma-globulin in complete Freund's adjuvant. Affinity was assessed by inhibition of plaque formation in the presence of free hapten. Anti-DNP PFC per 10(7) spleen cells were not diminished in short- and long-term R8% mice, and were increased in the former group at certain times after immunization. Affinity of indirect PFC was increased at Days 14 and 21 after immunization in short-term R8% mice and at Day 7 in R4% mice, and was similar in long-term R8% and N animals. No limitation in the heterogeneity of PFC affinities was observed in the restricted groups. Short-term restricted mice showed a rise of the high-affinity PFC subpopulation. The number of mice with hapten-augmentable PFC was diminished in the short-term R8% group at 7 days after immunization and in long-term restricted mice at 14 days, suggesting depressed levels of auto-anti-idiotypic antibodies in protein restriction.     

Serological and biological cross-reactivity of class II antigens between mice and humans in antigen-specific T-cell proliferative responses

Many studies have already been reported with regard to the serological cross-reactivities between the polymorphic determinants of murine Ia antigens and human HLA-DR antigens. In this paper, we examined the biological cross-reactivity of the polymorphism of Class II antigens in the xenogeneic antigen-presenting cell (APC)-T-cell interaction. The data indicate that purified protein derivative (PPD)-specific human T cells were not stimulated by PPD-pulsed murine APC from B10.S(9R) which possess I-As and I-Ek molecules serologically cross-reacting with human Class II antigens. On the contrary, B10.S(9R) T cells primed to PPD were stimulated by PPD-pulsed human APC. The failure of the murine APC-human T-cell interaction was not caused by the suppressive effect in culture with ongoing xenogeneic mixed lymphocyte reactions (MLR) or other cell culture conditions. Thus, a hierarchy of antigen-presenting ability in the xenogeneic APC-T-cell interaction was shown to exist.     

Splenic natural killer-cell activity in mice infected with Leishmania donovani

Several strains of inbred mice were infected with the protozoan parasite Leishmania donovani, and, at several points during the infection, spleens of groups of these mice were tested for natural killer (NK)-cell activity vs lymphoma target cells in vitro and were evaluated for parasite burdens. Generally, elevated followed by normal (compared to uninfected control mice) or subnormal NK responses occurred as the result of infection. Elevated NK responses were not accompanied by high circulating levels of interferon, yet infected mice responded to an injection of an interferon inducer with interferon production as great as control mice. No consistent correlations among susceptibility phenotype to L. donovani infection, spontaneous NK activity phenotype, and infection-induced NK activation/depression patterns were detected among the various strains of mice.     

Mechanisms of depression of splenic natural killer cell function in C57BL/6 mice infected with Leishmania donovani

C57BL/6 mice chronically infected with the protozoan parasite Leishmania donovani exhibit profoundly depressed splenic natural killer (NK) cell activity as measured by in vitro cytolysis of lymphoma target cells. Injection of infected mice with an interferon (IFN) inducer or in vitro treatment of infected splenocytes with IFN, a phorbol ester, or indomethacin failed to restore their NK activity to the degree shown by age-matched, uninfected mice. Fractionation of infected splenocytes by nylon wool, Sephadex G-10, or carbonyl iron and magnetism treatments was also unable to effect an increase in NK activity. Addition of infected splenocytes to uninfected ones in in vitro NK assays suppressed the NK activity of the latter, and the suppression could be partially or wholly abrogated by prior fractionation of infected splenocytes by the methods noted above. In vitro treatment of infected splenocytes with concanavalin A revealed the presence of NK activity in these cell populations. The results indicate that splenocytes in L. donovani-infected mice become insensitive to IFN stimulation; and the impairment of another, possibly IFN-independent pathway of NK-cell activation may also contribute to the observed L. donovani-induced depression in splenic NK activity in C57BL/6 mice.     

The production of T-cell growth factor (TCGF) in vivo in sheep

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000–20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.     

Suppressor T-cell hybridomas that exhibit various capacities to negatively regulate PFC responses in a T-cell dependent mouse model

Splenic leucocyte effector cells from 3-month-old C3H/HeJ mice which were antibody negative for influenza A virus were found to be spontaneously cytotoxic for mouse L929 fibroblasts infected with influenza virus, but not for uninfected cells. The cytotoxic activity was significantly enhanced if the effector cells were preincubated with splenic leucocytes from older 6- or 9-month-old antibody-negative mice before being added to the target cells. Enhancement appeared to be due to a cooperative effect between leucocytes from mice of different ages. Preincubation of effector cells with autologous sera markedly suppressed spontaneous cytotoxicity, but the suppression was significantly less with sera from 7- or 10-month-old mice than with sera from 3-month-old mice. Most cytotoxic activity was observed with a nylon wool-adherent subpopulation of leucocytes, although some cytotoxicity remained in the non-adherent fraction. The results suggested that spontaneous cytotoxicity was due to two subpopulations of cells, possibly adherent monocyte-macrophages and nonadherent natural killer cells.     

Glycerate kinase from leaves of C3 plants

D-Glycerate-3-kinase (EC 2.7.1.31) in six C3 species, including dicots (Pisum sativum, Spinacea oleracea, Antirrhinum majus) and monocots (Secale cereale, Hordeum vulgare, Avena sativa), ranged in activity from 44 to 353 mumol X mg chl-1 X h-1. Studies with protoplast extracts of these species indicate that the enzyme is localized in the chloroplasts. Glycerate kinase was partially purified from Secale (rye, 288-fold) and Pisum (pea, 252-fold) chloroplasts by DEAE-cellulose chromatography, sucrose gradient centrifugation, and chromatofocusing. The enzymes from both species showed similar physical (Mr = 41,000, pI = 4.6-4.7) and kinetic (Km ATP = 655 to 692 microM, Km D-glycerate = 180-188 microM) properties. Activity of the enzyme was essentially insensitive to variations in assay pH from 6.4 to 9.0 and to energy charge variations from 0.4 to 1.0. Rye glycerate kinase was able to utilize UTP and GTP but less effectively than ATP. Neither ADP nor pyrophosphate served as an energy source. Mn2+, Co2+, Ca2+, and Sr2+ could function as metal cofactors, although to a lesser extent than Mg2+. Millimolar levels of sulfate were found to significantly inhibit the enzyme while similar concentrations of other anions (Cl-, NO-3, NO-2, and acetate) had little or no effect.     

Pyruvate dehydrogenase activity in several tissues of genetically obese hyperglycemic mice

The active form (PDHa) and total activity of pyruvate dehydrogenase (PDH) were measured in homogenates from heart muscle, epididymal fat pads and liver of genetically obese hyperglycemic mice and compared with similar data derived from lean controls or Swiss albino mice. Both PDHa and total PDH activities were similar in heart muscle from all mice with a precipitous decrease in the PDHa upon fasting. Adipose tissue and liver of obese mice had a PDHa level that was almost two-fold higher than either lean control or Swiss albino mice. Fasting for 24 hours decreased the elevated activity of PDHa in adipose tissue and liver in obese mice to a value that was comparable to lean control or Swiss albino mice, fasted similarly. The elevation in both the active form and total activity of pyruvate dehydrogenase in livers from obese mice could explain the increased provision of acetyl-CoA units necessary for the accelerated hepatic lipogenesis observed with this mouse, a model for human obesity and insulin resistance.     

In vitro histamine H2-antagonist activity of the novel compound HUK 978

Histamine stimulated adenylate cyclase from guinea-pig fundic mucosa and 3H-tiotidine binding in guinea-pig cerebral cortex were used to assess the in-vitro histamine H2-activity of the novel H2-antagonist HUK 978. The results showed that HUK 978 was a more potent H2-antagonist than either cimetidine or ranitidine. HUK 978 was also shown to be devoid of activity at the histamine H1-receptor, the muscarinic receptor and the alpha and beta-adrenergic receptors.     

3H]Captopril binding to membrane associated angiotensin converting enzyme

[3H]Captopril binding to membrane fractions of rat tissues is saturable and reversible with a KD of 2.4 nM. [3H]Captopril binding and angiotensin converting enzyme measured with hippuryl-L-histidine-L-leucine are distributed in parallel between different tissues and brain regions, with highest levels in the choroid plexus, lung and corpus striatum. Captopril, N-(1(S)-carboxy-3-phenyl-propyl)-L-alanyl-L-proline, N-(1(S)-carboxy-3-phenyl-propyl)-L-lysyl-L-proline, teprotide, thiorphan and S-acetylcaptopril each have similar potencies for inhibition of [3H]captopril binding and of angiotensin converting enzyme. These data strongly indicate that [3H]captopril binds selectively to angiotensin converting enzyme. [3H]Captopril binding evaluation should help clarify the localization and function of angiotensin converting enzyme and assist in defining pharmacologic actions of captopril.     

Lectin-like activity from Persea americana

An extract from the seeds of Persea americana possessed an erythro-agglutinating activity. The agglutinin was devoid of specificity for carbohydrates, but interacted readily with basic proteins or basic polyamino acids. The interaction between the agglutinin and egg-white lysozyme was not inhibited by chaotropic salts, but was sensitive to relatively low concentrations of urea. An affinity chromatographic procedure was developed in an effort to purify the agglutinin. Products from the chromatographic procedure were found not to contain higher specific agglutinating activities than the crude extract. Amino acid acid analyses of the extract showed the presence of relatively high proportions of glutamic and aspartic acids. In addition, the extract contained phosphorus and a visible chromophore. The agglutinin was resistant to detergents and denaturants, and proteases, nucleases, and other enzymes. The results suggest that, as opposed to other plant agglutinins, the active component from Persea is not a protein. Similarly, in contrast to many lectins, the agglutinin from Persea was not mitogenic for mouse lymphocytes. The agglutinin partially inhibited the mitogenesis of lymphocytes when the cells were treated with concanavalin A, or with bacterial lipopolysaccharide.     

Anti-secretory activity of the novel H2-antagonist, HUK 978, in rat, guinea-pig and dog

The anti-secretory activity of the competitive H2-antagonist HUK 978 was determined in rat, guinea-pig and dog. In all systems examined, HUK 978 was more potent than cimetidine and ranitidine both intravenously and orally. In addition, the compound at approximately equipotent doses as these established H2-antagonists exhibited a significantly longer inhibitory profile following oral and systemic administration. Data from these pharmacological studies and the in vitro investigations previously reported, suggest that HUK 978 is a highly specific H2-antagonist and inhibits acid secretion for longer periods than other competitive compounds.     

Modulation of natural killer activity in mice following infection with Listeria monocytogenes

Mice that received a sublethal, intraperitoneal dose of viable Listeria monocytogenes, virulent strain 10403, exhibited a systemic increase in natural killer (NK) activity. The kinetics of the response differed with respect to the various effector cell populations analyzed. Resident peritoneal cells and peripheral blood leukocytes demonstrated high NK activity on Days 3, 7, and 10. Peak spleen and bone marrow NK activity was observed on Day 3, returning to normal levels by Day 7. In contrast, peritoneal exudate cells, elicited with proteose peptone, expressed enhanced NK activity for 60 days following infection with viable Listeria. Augmented NK activity was detected with all cell types as early as 12 hr after infection. The intraperitoneal injection of nonviable antigenic preparations derived from L. monocytogenes, strain 10403, resulted in the enhancement of peritoneal and splenic NK activity. In contrast, mice that received an intraperitoneal injection of avirulent Listeria, strain 19113, failed to express enhanced levels of NK activity. The genetic trait of anti-listerial resistance which is associated with non-H-2 linked genes was of no importance with respect to enhanced NK activity. Listeria-resistant C57BL/6J and Listeria-susceptible DBA/2J mice both produced systemic augmentation of NK activity following infection. NK activity was not abrogated by macrophage depletion or by treatment with anti-Thy 1.2 serum plus complement. These results confirm the potent immunostimulatory capacity of virulent Listeria for NK activity and provide further insight into the kinetics of this response in various lymphoid compartments. The protracted augmentation of NK activity of elicited peritoneal exudate cells as compared to nonelicited peritoneal cells in Listeria-primed mice suggests that the influx of inflammatory cells may provide NK-enriched and/or accessory populations for immunopotentiation of NK activity in inflammatory sites.     

Identification of [3H] histamine binding sites on gastric mucosal cells unrelated to histamine H2-receptors

Acidic unfolding process of myoglobin was investigated in the presence of external ligands (azide, cyanide, fluoride and imidazole). With azide, cyanide and fluoride as ligand, myoglobin unfolds through a single exponential decay process, whereas it is not the case with imidazole. No faster decays were observed as in the case of myoglobin without external ligands. These results demonstrate the important role of iron-ligand interaction on the conformational stability of myoglobin.