Guinea Pig Lymph Node Research Articles

Histological appearance of the site of inoculation and lymph nodes of guinea-pigs at various times after infection with Leishmania enriettii

An investigation was undertaken to determine the immunological response of guinea-pigs to Leishmania infection through observation of the histology at the site of infection and regional lymph nodes of immune and normal animals at various intervals following inoculation with L. enriettii. Determinations of the leishmania growth-inhibitory activity of the animals' plasma were made concurrently. One day following challenge the immune guinea-pigs showed a marked inflammatory response at the site of infection whereas the normals did not. In the immune animals, the cell population at the infection site consisted of macrophages, plasma cells, eosinophils and lymphocytes, while in the normal animals, macrophages were the predominant cell type observed. Cell infiltration in the paracortical areas of the draining lymph node started on the third day and gradually increased until the sixth week of infection. A gradual and steady development of the germinal centres in the draining lymph nodes of the infected guinea-pigs began after the second week and reached a maximum after the sixth to eighth week of infection. Methyl green pyronin staining of the lymph nodes demonstrated many large pyroninophilic cells in the germinal centres and a few in the paracortical area. The leishmania growth-inhibitory factor was first detected in the plasma on the 44th day after infection and increased up to the 60th day.

Eriochrome black, a means for reduction of nonspecificity in immunofluorescence.

Fluorescent preparations with a high degree of nonspecific background staining were treated with a 1 : 60 or 1 : 30 (w/v) aqueous solution of Eriochrome black for 10 sec and then remounted. Nonspecificity was thereby considerably reduced. The technique was applied to cryostat sections of guinea pig lymph nodes containing <i>Serratia marcescens </i>and <i>E. coli, </i>with tissues of mice suffering from a pneumococcus septicemia, with Brucella-positive bovine placentas and with brain impressions of rabid foxes, mice and a cat. In all the systems studied, the fluorescent conjugate was purified only by gel filtration through Sephadex G-50, a further fractionation by ion exchange chromatography was shown to be superfluous.

Suppression of In Vitro Antigen-Induced Lymphocyte Transformation by Carrageenan, a Macrophagetoxic Agent

Abstract Carrageenan, a high molecular weight sulfated polygalactan with known macrophage toxic properties was added to cultures of normal and sensitized lymphocytes from human peripheral blood and guinea pig lymph nodes to determine its effect on phytohemagglutinin, and antigen-induced 3H-TdR lymphocyte incorporation. Carrageenan was not toxic for guinea pig or human lymphocytes per se and induced no lymphocyte transformation in vitro. With high doses in the range of 1000 µg/ml and above, some decrease in 3H-TdR uptake was noted. Phytohemagglutinin-induced human and guinea pig lymphocyte transformation was not affected by carrageenan but a marked dose-dependent suppression of antigen-induced 3H-TdR lymphocyte uptake was noted in both species. This suppressive effect was not due to reagent or antigen dilution. It was observed when carrageenan was added to cell cultures prior to antigen or up to 6 hr later, but not when added after 17 hr. These findings suggest that carrageenan acts at an early stage of macrophage-antigen processing and are consistent with previous reports of inhibition of antigen-induced lymphocyte transformation using anti-macrophage serum. They provide further evidence that phytohemagglutinin-induced lymphocyte transformation is not mediated by macrophages and that macrophage-lymphocyte interaction occurs with cells from the peripheral blood as well as from lymph nodes.

Further Studies on Migration Inhibitory Factor (MIF): Evidence for its Glycoprotein Nature

Abstract Migration inhibitory factor (MIF) obtained from sensitized guinea pig lymph node lymphocytes that had been stimulated by specific antigen (o-chlorobenzoyl-bovine γ globulin) was characterized by enzymatic treatment and isopycnic centrifugation in a CsCl density gradient. MIF incubated with water-insoluble chymotrypsin lost its biologic activity. Further, neuraminidase, which hydrolyzes terminal sialic acid, abolished MIF activity. When MIF was centrifuged in CsCl density gradients, it had a buoyant density (ρ25 = 1.430) slightly greater than that of the protein reference substance (ρ25 = 1.352), which is consistent with its being a glycoprotein. The results indicated that MIF is a glycoprotein and that sialic acid is necessary for its biologic activity.

DNA replication in antigen stimulated guinea-pig lymph node cells.

Modifications in the pattern of DNA synthesis after antigenic stimulation may reflect an alteration in the sequence of gene replication triggered by antigen.

Studies on migration inhibitory factor (MIF): Recovery of MIF activity after purification by gel filtration and disc electrophoresis

Migration inhibitory factor (MIF) obtained from sensitized guinea pig lymph node lymphocytes stimulated by specific antigen ( o-chlorobenzoylbovine gamma globulin) in the absence of serum was characterized by Sephadex G-100 gel filtration, sucrose gradient centrifugation, and disc electrophoresis. Peak MIF activity was found after Sephadex gel filtration or sucrose gradient centrifugation in fractions containing molecules slightly smaller than albumin, indicating a molecular weight range of 35–55,000. No MIF activity was found in fractions containing molecules the size of immunoglobulins or in the fractions containing small molecular substances, such as lysozyme. A method was developed for preparing acrylamide gels from which material could be eluted which is nontoxic to cells in culture. When MIF-rich Sephadex fractions were further fractionated by disc electrophoresis at pH 9.1, peak MIF activity was consistently eluted from the fraction containing molecules migrating anodally to albumin. The three procedures employed demonstrated that MIF activity could be separated from albumin on the basis of size and charge.

Cell dynamics in phytohemagglutinin (PHA) stimulated guinea-pig lymph node lymphocyte cultures.

Cell dynamics in phytohemagglutinin (PHA) stimulated guinea-pig lymph node lymphocyte cultures.

Phytohemagglutinin-induced transformation and aggregation of guinea pig lymphocytes.

Mitogenic agents which cause a high percentage of blast transformation when added to short-term lymphocyte cultures also cause marked cell aggregation. Conditions which promote cell-to-cell contact may increase the blastogenic reaction as evidenced in antigen-stimulated lymphocyte cultures (1, 2). Removal of those cells capable of adhering to glass eliminates the transformation seen in antigen-stimulated cultures (1–3). Despite the evidence that cell contact is an important aspect of response to specific antigens, the relationship of marked aggregation seen with phytohemagglutinin (PHA) and other nonspecific mitogens to blastogenesis is less clear. Lymphocytes purified by the glass bead method still transform when treated with PHA (1, 2). Coulson (4) has observed PHA-induced transformation even when cells are embedded in agar prior to treatment so that cell contact is eliminated. In the agar system it would seem difficult to measure the magnitude of this response. In the present work, the response of guinea pig lymph node lymphocytes to PHA is related to the aggregation characteristics of the treated cells. Materials and Methods. Hartley strain inbred guinea pigs (Camm Research Institute, Inc., Wayne, N.J.) 300–350 g males were used in all experiments. These animals were immunized by subcutaneous injection into each hind footpad, 0.2 ml of alumprecipitated DPT antigen (Tri-Solgen, Eli Lily & Co.) at 10-day intervals. Ten to 20 days after the second immunization, the animals were sacrificed. Under aseptic conditions, popliteal lymph nodes were collected in TC 199 (Difco) with 20% fetal calf serum, gently minced, and expressed through a 100-gauge stainless steel wire mesh. This yielded a 98% single cell suspension which was washed twice and adjusted to a concentration of 4 × 106 cells/ml. A 0.5-ml portion of this suspension was then added to 1.5 ml of TC 199, with or without PHA (control), and cultured in vertical, tightly stoppered 15-ml round bottom Kimax tubes at 37° in 5% CO2-air. Cells were routinely harvested at 1, 48, and 96 hr. The PHA used in all experiments was from a lot prepared in this laboratory by the method of Börjeson et al. (5), contained 2.8 mg of protein/ml, and was stored frozen at —20°. A new vial was opened for each experiment. The optimum dose of PHA for a culture containing 1 × 106 cells/ml was 0.001 ml (2.8 μg of protein) /ml of culture.

Stress effect of antilymphocyte serum.

Antilymphocyte serum prepared in the rabbit against guinea-pig lymph node cells produces a sustained weight loss when injected into normal guinea-pigs over 14 days, compared with a very transient loss produced by normal rabbit serum. Antilymphocyte serum also produces a much greater increase in plasma cortisol level than normal rabbit serum. Part of this effect may be due to adrenal stimulation, and further studies of its activity in adrenalectomized animals are suggested.

A quantitative study of the stimulation of DNA synthesis in lymph node cell cultures by anti-lymphocyte serum, anti-gamma globulin serum, specific antigen, and phytohemagglutinin.

Rabbit anti-guinea pig lymphocyte serum is an efficient stimulus of the synthesis of DNA by guinea pig lymph node cells in vitro. The ability of ALS to stimulate lymphocytes is characterized by its lack of dependence on prior sensitization, the magnitude of the response it elicits, and the stimulation of all sensitive lymph node cells simultaneously within a very narrow range of ALS concentrations. In contrast to this homogeneous response to ALS, the stimulation of lymph node cells by antigen proceeds in graded fashion over a wide range of concentrations, thus reflecting the heterogeneity of the response of sensitized cells to antigen. PHA gives a response which is intermediate between that of ALS and antigen. ALS appears to have specificity for membrane determinants shared by lymphocytes but not found on other tissues. This specificity does not involve cell-bound gamma globulin. The serum activity mediating lymphocyte stimulation as well as cytotoxicity is readily removed by absorption with lymph node cells.

Protein synthesis in lymph nodes during the development of contact sensitivity in the guinea-pig

3rotein synthesis in lymph nodes of non-sensitized guinea-pigs and of guinea-pigs sensitized 4 and 6 days previously to the chemical agent ‘oxazolone’ was studied by in vitro incorporation of 14C-leudine. Analysis of extracts of these lymph nodes by DEAE-cellulose chromatography and autoradio-immunoelectrophoresis showed that there was a marked increase in 14C-leudine incorporation by lymph nodes on the fourth day after sensitization and a subsequent reduction by the sixth day. Correlated with this, there was a significant increase in synthesis of γ 2-globulin by 4-day sensitized lymph nodes which were also synthesizing a number of other proteins, at least 3 of which migrated electrophoretically in the α-globulin region. Radioactive peaks from DEAE-cellulose chromatography of extracts of 4-day sensitized lymph nodes were sub-fractionated by Sephadex G-200 gel-filtration. This revealed a number of newly-synthesized components which were not detectable by immunoelectrophoresis. The increased synthesis of proteins at 4 days after sensitization coincides with the presence in the lymph node of maximum numbers of immunoblasts, and the possible significance of this is discussed.

Cellular Activities in Hypersensitive Reactions

Summary Small lymphocytes from lymph nodes of guinea pigs with tuberculin hypersensitivity react specifically with tuberculoproteins, as revealed by fluoresceinated guinea pig antituberculoprotein serum. Fluorescence occurs as a rimming or halo effect in 5 to 20% of such cells. Occasional glowing cells are seen in normal cell preparations, at a lower level of intensity of fluorescence. Although the methods employed suggest that antibody globulin adsorbed to cells, or produced by them, is not implicated in the reactive property, this possibility cannot be excluded.

Studies in Experimental Eosinophilia

Summary The draining lymph nodes of guinea pigs were examined histologically after the injection of BSA or hemocyanin into footpads. When a 10-mg dose of hemocyanin was administered, increased numbers of eosinophils were detected as early as 4 hr, and the peak response was seen at 12 hr. Margination, diapedesis and perivascular cuffing by eosinophils were prominent features in lymph nodes removed during the 1st hr, and such changes were evident as early as 5 min after the first exposure to hemocyanin. Similar findings were obtained with BSA, but the earliest changes were noted at 1 hr. When BSA, labeled with fluorescein, was injected into footpads, green fluorescence was noted in the draining lymph node as early as 5 sec. The experiments are consistent with the view that de novo synthesis of antibody may occur within 5 min of the first encounter of an organism with an antigen.

An Anti-Mycobacterial Principle of Peritoneal Mononuclear Cells

Summary A factor inhibiting the growth of tubercle bacilli was found in disrupted peritoneal mononuclear cells from normal and BCG-immunized guinea pigs. It was shown that mononuclear cells from immunized guinea pigs were, on the average, 4 times as active as were cells from normal animals. However, great variability existed among individual guinea pigs immunized with BCG with respect to yield of inhibitory material. Serum did not increase the action of the cell-bound inhibitor but, in some instances, seemed to neutralize its action. The growth inhibitor proved to be directed against tubercle bacilli only, and other facultative intracellular organisms, such as Brucella or Salmonella, were not inhibited when tested under similar conditions. Delayed hypersensitivity to tuberculoprotein and yield of anti-mycobacterial principle of mononuclear cells from BCG-immunized guinea pigs or animals injected with complete Freund's adjuvant were unrelated. Cells obtained from guinea pig lymph nodes or spleen contained on a per cell basis less antimycobacterial substance than peritoneal mononuclear cells, whereas lysed cells from guinea pig liver or kidney proved to be highly inhibitory for tubercle bacilli.

Experimental eosinophilia. VII. Lymph node responses to altered gamma globulins

The production of inflammatory cell infiltrations within popliteal lymph nodes regional to challenged subcutaneous foot pad sites served as an experimental model for evaluating possible eosinotactic stimuli in the guinea pig. The following were studied: purified native serum gamma globulins of bovine (BGG), rabbit (RGG), and guinea pig (GPGG) origins; chemically altered gamma globulins in aggregated states (AgBGG, AgRGG, AgGPGG) and in urea-treated denatured forms (DeBGG, DeRGG); preformed soluble BGG antigen-antibody complexes; and BGG antigen-rabbit antibody systems in passive sensitization procedures. Cellular responses were evaluated by microscopic study of lymph node smear preparations and by histologic sections. Significant degrees of eosinophil granular leukocyte infiltrations were found in the sinuses of lymph nodes of guinea pigs receiving AgGG of both foreign and homologous species origins, DeBGG and DeRGG, soluble antigen-antibody complexes, and of those subjected to antigen-antibody reactions. Lesser degrees of involvement demonstrated in animals receiving native gamma globulins were attributed to the content of altered forms in these preparations. Specific in vivo antigen-antibody reactions were additionally characterized by associated polymorphonuclear granular leukocyte responses within cell infiltrates. The emergence of eosinophilia did not depend upon any potential associated role for the inflammatory stimulus as an immune reactant. These findings suggest that eosinotactic influences of antigen-antibody union may be related to the presence of antigen-antibody complexes in the form of molecular aggregates.

THE CROSS-REACTIVITY AND TRANSFER OF ANTIBODY IN TRANSPLANTATION IMMUNITY.

A close inomunochemical relationship of Forssman-type antigens in cells or in a methanol extract of guinea pig kidneys, lymph nodes, and platelets, of horse kidneys, and of sheep erythrocytes was demonstrated by complement-fixation, agglutination, and inhibition of hemolysis. The dissociation of antibody from several cross-reacting complexes and re-association with antigens of erythrocytes used for immunization was inferred from quantitative hemolytic assays. This preferential affinity of antibody for the antigen used for immunization is proposed as an immunochemical model for reactions which function in graft rejection phenomena wherein the donor and recipient tissues share cross-reacting antigens.

A vascular permeability factor extracted from normal and sensitized guinea-pig lymph node cells

Extracts of guinea-pig lymph node cells produced a striking increase in the vascular permeability of rat skin. The active principle has been differentiated from histamine, hydroxytryptamine, bradykinin, Substance P and salivary kallikrein.

The morphology of the active and inhibitory phases of immunity in guinea pigs immunized with heated paratyphoid vaccine

In repeated multiple administrations of heated B. paratyphi A vaccine certain morphological changes took place in the lymph nodes (especially regional) of guinea pigs. These consisted of rapid reduction of the total number of plasmatic cells, especially of the young forms, and of the total number of reticular cells, accompanied by a marked atrophy of reticular tissue.

The use of specific "lymphocyte" antisera to inhibit hypersensitive reactions of the "delayed" type.

Rabbit antisera against normal guinea pig lymph node, when injected into guinea pigs, produced transient depression of the level of blood lymphocytes. It had no effect on other circulating cellular elements. Repeated injection over several days produced lymphopenia, which became progressively less marked with continued treatment, and clear-cut depletion of small lymphocytes in lymph nodes, whether draining an inoculation site or remote. In guinea pigs treated with lymphocyte antiserum, there was marked suppression of the tuberculin and contact allergic reactions and the "delayed" skin reaction to purified diphtheria toxoid, and a relative suppression of allergic encephalomyelitis and the rejection of first set skin homografts. There was a slight effect on second set graft rejection and no effect on PCA or the reversed passive Arthus reaction. Non-specific reactions to intradermal turpentine or to concentrated dinitrochlorobenzene placed on the skin were moderately reduced. The suppression of these reactions (except allergic encephalomyelitis) was closely correlated with the degree of lymphopenia. Lymphocyte antiserum absorbed with normal blood white cells lost both its lymphopenic effect and its ability to suppress the tuberculin reaction. It is tentatively concluded that a circulating mononuclear cell, probably the small lymphocyte, is the primary reactant in the various types of delayed hypersensitive reactions.

Comparison of lymphoid cells of bone marrow and lymph nodes of rabbits and guinea pigs

Comparison of lymphoid cells of bone marrow and lymph nodes of rabbits and guinea pigs