Conditions of the formation of pyrogenic substances by delayed hypersensitive cells in contact with antigenin vitro

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   В ряде систематических оплтов дыли подробно определены биологические условия возникновения пирогенных веществ при реакции гиперсензитивных клеток со специфическим антигеном in vitro, влияние количества клеток и антигена, температуры и продояжительности инкудации, жизнеспосодности клеток и т. п.     
   
   

Influence of supramammary lymph node extract on in vitro cell proliferation

Abstract.   Objectives: Experiments were conducted to evaluate whether or not bovine supramammary lymph node extract (LNE) could support cell proliferation when it was substituted for bovine growth serum (BGS) in cell culture media. Materials and Method s: Two different preparations of LNE were tested. The first yielded protein concentration of 3 mg/mL and the second contained 27 mg/mL protein. Three cell lines (MDA-MB-435, MAC-T and 1C6) were used in serum starvation assays to evaluate LNE. Cell proliferation assays were used to determine growth stimulation in the presence of LNE, and short-term or rapid adaptation cultures were evaluated for LNE effects on cell survival. Results : Heat-inactivated preparation 1 supported cell proliferation as well as or better (12–39%) than BGS following 2 days of serum starvation in culture. The second lymph node preparation provided a stimulatory effect (263–702% greater than BGS across all cell lines) following serum starvation at 2.7 and 5.4 mg/mL protein supplementation. A gradual adaptation process with lymph node supplementation into media maintained cell population growth on a short-term basis. However, once cells were trypsinized or scraped and re-seeded into 2.7 mg/mL LNE protein containing media, cells were unable to re-adhere, leaving them detached, and eventually appearing to be dead. Conclusion s: Substitution of BGS with LNE protein dramatically stimulated cells to proliferate, but did not allow for rapid cell population growth adaptation in vitro .     

Antigen-specific lysis in vitro of lymph node cells of intact mice injected with RNA from viable lymph node cells of immunized animals]

Supernatant fluid obtained after centrifugation of the suspension of viable lymph node cells of immunized animals proved to induce in vivo in the lymph node cells of intact mice sensitivity to lysis with a specific antigen in vitro. This property was possessed after chromatography of the supernatant fluid on Sephadex G-200 by the 3rd fraction (MW about 30000 dalton). DNA-ase, trypsin or deproteinization failed to influence whereas RNA-ase inactivated this fraction in respect to the inducing properties.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

Development of IgE-forming cells in vitro from rat mesenteric lymph node cells.

Mesenteric lymph node cells from normal rats and rats infected with Nippostrongylus brasiliensis (Nb) were cultured with pokeweed mitogen (PWM) or Nb antigen, and the development of IgM-, IgG2a-, or IgE-containing cells was assessed by immunofluorescence. Normal lymph node cells stimulated with PWM developed into both IgM- and IgE-containing cells, whereas similar stimulation of cells from Nb-infected rats resulted in the development of IgM-, IgG2a-, and IgE-containing cells. The in vitro plasma cell response to PWM was dependent on the presence of T lymphocytes. Lymph node cells from Nb-infected rats responsed to Nb antigen and developed into plasma cells of IgM, IgG, and IgE classes. The response was antigen specific and required antigen-primed T cells. Depletion of IgE-bearing cells or IgM-bearing cells before stimulation with either PWM or Nb antigen diminished the level of IgE forming cell development, suggesting that IgE-IgM double bearing cells are precursors of IgE-forming cells. The distribution of the three isotypes among the If-forming cells that developed in response to PWM was influenced by the source of both B and T cells. When B cells from Nb-infected rats were employed as a source of precursors, T cells from infected animals were more effective than normal T cells for the development of IgE-forming cells, whereas the latter cells were more effective for the development of IgG2a-forming cells than T cells from infected animals.     

Induction of lymph follicle formation with several mitogens and adjuvants in the mouse popliteal lymph node

The formation of lymph follicles in draining popliteal nodes was investigated in young adult male mice which had been injected in the rear footpad with several mitogens and adjuvants, and killed after 3-21 days. PPD (100 micrograms-1 mg) and PHA (25-500 micrograms) induced germinal centers in association with existing follicles and mild plasmacytosis, but failed to produce new follicles in draining nodes. Endotoxin LPS (50-200 micrograms), Con A (50 micrograms-1 mg) and PWM (50 micrograms-1 mg) induced germinal centers within existing follicles and plasmacytosis, and also produced new follicles which soon developed germinal centers. Both Freund's complete and incomplete adjuvants (FCA and FICA, 25 microliters) induced virtually no germinal centers and plasmacytosis, but produced a significant number of new primary follicles. Poly (A, U) (600 micrograms) produced neither germinal centers nor plasmacytosis, and did not induce new follicles. Analysis of the distribution of lymphoid cells which had incorporated 3H-thymidine in the draining nodes at 3 days after the injection of test substances indicated that PPD, PHA, LPS, Con A and PWM preferentially stimulated in vivo the same types of lymphocytes as they do in vitro. FCA triggered lymphocyte activation in the deep cortex, whereas Poly (A, U) appeared not to stimulate lymphocytes in vivo. In further experiments, induction of lymph follicles with artificially precipitated PPD and PHA was studied. The draining nodes treated with alum-precipitated PPD or PHA were found to produce a significant number of new follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific interaction of phagocytosing mononuclears with T cells during formation of the thymocyte-induction factor

Data are provided on specific secretion by macrophages of a factor inducing the functional differentiation of thymocytes. The contact of mononuclear phagocytes with lymphocytes of T-cell type induces the formation of this transmitter. Interaction of macrophages with B-cells does not lead to the secretion of this monokine. Treatment of macrophages with nonspecific stimulators exerts no effect on the production of the factor.     

The interaction of [3H]TPA with bovine lymph node lymphocytes in vitro

The potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), is an effective modulator of DNA synthesis in bovine lymph node lymphocytes in culture. The effect of TPA on cellular proliferation depends to a great extent on the length of exposure and the mitogenic stimulus. Addition of TPA along with mitogenic lectins enhances DNA synthesis. Exposure to TPA for 2 days before addition of lectins depresses DNA synthesis. There is evidence that some inhbitory material other than TPA is formed during the longer incubation. Therefore, in this study, we used [³H]TPA to determine (i) the amount of material retained by the cells after preincubation and (ii) if TPA was metabolized during this culture period. We found that after incubation with 10^?7 M, [³H]TPA, less than 3% of the radioactivity was retained by the cells. This was released by incubation in fresh medium. All of the cell associated material appeared to be TPA. However, the TPA in the medium was degraded by about 30% during a 2-day incubation to 12-O-tetradecanoyl-phorbol (TP), phorbol-13-acetate (PA) and phorbol. The source of the hydrolytic activity appeared to be serum because the same effect was seen in the absence of cells but was not seen in the absence of serum. These metabolites when added directly to the lymphocytes had no effect on DNA synthesis. Moreover, the amount of TPA retained by the cells and released into the medium was too small to account for the inhibitory activity of medium from TPA-treated cells. Studies are in progress to determine the nature of the inhibitory material after exposure to TPA.     

In vitro proteolysis of human plasma low density lipoproteins by an elastase released from human blood polymorphonuclear cells

In vitro incubation of human plasma low density lipoproteins (LDL) with human blood polymorphonuclear cells (PMN) for 1 h at 37 degrees C resulted in an increased (2-4-fold) release into the medium of an enzymatic activity which co-eluted with LDL by column chromatography at physiological ionic strength but dissociated from it in high salt media in an ultracentrifugal field. The release of this enzymatic activity increased with increasing concentration of LDL in the medium and caused the hydrolysis of the LDL apoprotein B100 as indicated by the appearance of 7-8 low molecular weight bands (immunoreactive with anti-LDL) which were not present in the electropherogram of control LDL. The proteolytic activity was identified as an elastase by the following criteria: 1) capacity to hydrolyze the synthetic substrate methoxysuccinyl-Ala-Ala-Pro-Val-4-methylcoumaryl-7-amide known to be specific for the PMN elastase, 2) pattern of apo-B proteolysis identical to that exhibited by pure PMN elastase, 3) inhibition of the proteolysis by the elastase inhibitor methoxysuccinyl-Ala-Ala-Pro-Val-CH2Cl, 4) identity in molecular weight (28,000-30,000) of this activity with a pure preparation of PMN elastase labeled with [3H]diisopropylfluorophosphate. Based on thiobarbituric acid analyses and the lack of effect by vitamin E, oxidative events appeared to play no detectable role in apo-B proteolysis. Since we previously reported (Byrne, R. E., Polacek, D., Gordon, J. I., and Scanu, A. M. (1984) J. Biol. Chem. 259, 14531-14543) that high density lipoprotein-3 promotes the in vitro release of PMN elastase which cleaves apo-A-II, it is apparent that in vitro, both LDL and high density lipoprotein, two of the major plasma lipoprotein classes, can affect the export from PMN of an elastase which exhibits proteolytic action on apo-B and apo-A-II.