Guinea Pig Lymph Node Research Articles

Bacillus Calmette–Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Comparison of the predictive nature of the Genomic Allergen Rapid Detection (GARD) assay with mammalian assays in determining the skin sensitisation potential of agrochemical active ingredients

Alternatives to mammalian testing are highly desirable to predict the skin sensitisation potential of agrochemical active ingredients (AI). The GARD assay, a stimulated, dendritic cell-like, cell line measuring genomic signatures, was evaluated using twelve AIs (seven sensitisers and five non-sensitisers) and the results compared with historical results from guinea pig or local lymph node assay (LLNA) studies. Initial GARD results suggested 11/12 AIs were sensitisers and six concurred with mammalian data. Conformal predictions changed one AI to a non-sensitiser. An AI identified as non-sensitising in the GARD assay was considered a potent sensitiser in the LLNA. In total 7/12 GARD results corresponded with mammalian data. AI chemistries might not be comparable to the GARD training set in terms of applicability domains. Whilst the GARD assay can replace mammalian tests for skin sensitisation evaluation for compounds including cosmetic ingredients, further work in agrochemical chemistries is needed for this assay to be a viable replacement to animal testing. The work conducted here is, however, considered exploratory research and the methodology needs further development to be validated for agrochemicals. Mammalian and other alternative assays for regulatory safety assessments of AIs must provide confidence to assign the appropriate classification for human health protection.

Cytomorphological characterization of lymphadenopathies in guinea pigs: study of 31 clinical cases

ABSTRACT Lymphadenopathy is a commonly found condition in guinea pigs that suffer diseases caused by bacterial organisms as Streptococcus zooepidemicus, Yersinia pseudotuberculosis, Bordetella, and Salmonellae (S. linate) which access cervical lymph nodes via an abraded oral mucosa. In this study, we investigate all possible alterations present in cytological smears obtained from cervical lymph nodes of guinea pigs with lymphadenopathy. Thirty-one clinical cases of guinea pigs with different grades of lymphadenomegaly and 10 clinically healthy guinea pigs were considered. The fine-needle nonaspiration cytology (FNNAC) provided an adequate quantity and quality of samples for cytological analysis. Air-dried FNNAC smears yielded excellent results with Wright stain. From 31 evaluated clinical cases, 64.5% were compatible with pseudoeosinophilic lymphadenitis, 22.6% compatible with reactive lymphatic node, 6.45% compatible with lymphoma, and 6.45% with Hodgkin's-like lymphoma. The findings revealed the usefulness of the FNNAC technique and Romanowsky stains as fast and easy methods for lymph node sampling and analysis, and also, the importance of recognizing various causes of lymphadenopathies associated with morbidity and mortality in guinea pigs which would influence treatment protocols and prognosis for patients.

Evolution of the Serendipitous Discovery of Macrophage–Lymphocyte Interactions

Evolution of the Serendipitous Discovery of Macrophage–Lymphocyte Interactions

Antilisterial activity of lactic acid bacteria against Listeria monocytogenes strains originating from different sources

Eight individual bacteriocin-producing lactic acid bacteria (LAB) strains and three bacteriocin-non-producing cheese starter cultures were evaluated for their ability to inhibit the growth of six Listeria monocytogenes strains, originating from the guinea-pig lymph nodes, raw cow milk, and manufacturing dairy equipment. Results showed that either live cells or cell-free neutralised supernatant (CFNS) and/or heated CFNS of six individual LAB strains (Lcc. lactis subsp. lactis CCDM 416 and NIZO R5, Lbc. plantarum HV 11 and DC 1246, P. acidilactici HV 12, and Ent. mundtii CCM 1282) and one starter culture (DELVO-ADD<sup>®</sup> 100-X DSF) were effective in the suppression of at least one listeria strain. Neither any individual LAB strain nor starter culture was antagonistic toward all studied L. monocytogenes strains, indicating diverse sensitivity/resistance among L. monocytogenes strains to antimicrobial compounds of LAB. The significant susceptibility of listerias isolated from raw milk and dairy equipment together with the strong antilisterial activity of DELVO-ADD<sup>® </sup>100-X DSF could be applied in dairy technology, where commonly used starter cultures could play both the biopreservative and fermentation role.  

Immune reactions of CD4- and CD8-positive T cell subpopulations in spleen and lymph nodes of guinea pigs after vaccination with Bacillus Calmette Guerin

Vaccination of guinea pigs with Mycobacterium bovis BCG confers partial resistance against infection with Mycobacterium tuberculosis. Induction of immunity is associated with a strong T cell response. The reactions of the cytotoxic and helper T lymphocyte subsets after BCG vaccination were analyzed by cytofluorometry and in functional tests. The relative number of CD8 + T cells in the spleen increased substantially after injection of BCG. In vitro restimulation after immunization induced a strong proliferative response but no cytotoxic reactions of CD8 + T cells against BCG-infected macrophages. A specific induction of IFN-γ and RANTES mRNA was observed after vaccination particularly in CD8 + but not in CD4 + T cells of the lymph nodes.

Accelerated influx of dendritic cells into the lymph nodes draining skin sites exposed to attenuated cercariae of Schistosoma mansoni in guinea-pigs.

We compared temporal changes in the cell composition of the skin-draining lymph nodes (SLN) in guinea-pigs exposed percutaneously to normal or attenuated cercariae of Schistosoma mansoni. Different populations were analysed by flow cytometry of double-stained cells by monoclonal antibodies to the major histocompatibility complex class II molecule and lymph node cells of guinea-pigs. Exposure to S. mansoni caused a marked increase in the proportion of B cells and dendritic cells (DC) on day 2, reaching a peak number on day 4. These changes were comparable in both infected and vaccinated animals exposed to normal or attenuated parasites, respectively. Total number of DC, however, showed different kinetics; in infected animals, the number peaked on day 2 and then gradually declined, whereas it reached a higher peak on day 4 in vaccinated animal. Daily injection of bromo-deoxyuridine after exposure to the parasite reduced the total number of DC in the SLN on day 4. A reduction in DC counts in the contralateral side SLN was also evident in vaccinated animals. Our results indicate that a significant number of newly formed DC are recruited to the skin by 4th day of vaccination, followed by increased efflux to the SLN. It is possible that retention of attenuated S. mansoni in the skin may cause accelerated recruitment of newly formed DC from the bone marrow, and facilitate transport and processing of antigens highly expressed on attenuated parasites.

Effect of pyrimidine derivatives on adenylate cyclase system of immunocompetent cell regulationin vitro

The pyrimidine derivatives xymedone and diucifon decrease the activity of adenylate cyclase, as shown in experiments on thymocytes and lymphocytes of guinea pig lymph nodes and human peripheral blood lymphocytes. Inhibition of the enzyme depends on the subpopulation and species appurtenance of immunocompetent cells. The relationship between the results and effects of pyrimidine derivatives in experiment and clinical setting is discussed.

Protein deficiency induces alterations in the distribution of T-cell subsets in experimental pulmonary tuberculosis.

Previous research has suggested that dietary protein deficiency alters resistance to experimental pulmonary tuberculosis, in part, by affecting the distribution and trafficking of antigen-reactive T cells. In this study, guinea pigs were maintained on either a protein-deficient (10% ovalbumin) or control (30% ovalbumin) diet and infected 4 to 6 weeks later with a low dose of virulent Mycobacterium tuberculosis H37Rv by the respiratory route. Monoclonal antibodies directed against the CD4 or CD8 markers on guinea pig lymphocytes were used in a flow cytofluorometric assay to determine the proportion of each subset in the peripheral circulation, spleen, and bronchotracheal lymph nodes at 4 weeks after infection. In uninfected guinea pigs, only the spleen exhibited an effect of diet on T-cell distribution, with small but consistent reductions in the proportions of both CD4 and CD8 T lymphocytes. However, following infection, protein deficiency exerted a profound effect on T-cell distribution. Malnourished, tuberculous guinea pigs harbored only 20 and 60% of the T cells (as a proportion of total lymphoid cells) found in the spleen and blood, respectively, of their well-nourished counterparts. Normal relative proportions of CD4 and CD8 cells were observed, however. In striking contrast, the bronchotracheal lymph nodes of protein-deprived guinea pigs with tuberculosis contained more than twice the numbers of T cells of control guinea pigs, and the normal CD4-to-CD8 ratio was reversed. Peripheral T-cell function, as measured by the delayed hypersensitivity skin test to tuberculin, and antigen-induced lymphoproliferation in vitro were markedly suppressed in protein-malnourished animals. Conversely, purified protein derivative-induced (but not concanavalin A-induced) proliferation was significantly enhanced in cultures of lymph node cells from protein-deprived tuberculous animals. Taken together, these results suggest that immunological abnormalities and loss of antimycobacterial resistance in the lungs of protein-deficient guinea pigs may be explained, in part, by sequestration of antigen-reactive T cells in the lymph nodes draining the site of infection.

Antigen Stimulation Alters the Expression of S-100 Protein in the Giant Macrophages and Follicular Dendritic Cells of the Guinea Pig Lymph Nodes

Injections of chick erythrocytes were employed to examine the relationship between expression of S-100 protein and antigen stimulation in the giant macrophages (GM) and follicular dendritic cells (FDC) of guinea pig lymph nodes by an immunocytochemical method. Popliteal lymph nodes were observed 1, 3, 6 and 12 h, then 1, 2 and 3 days, and finally 1, 2 and 4 weeks after injection of chick erythrocytes into hind foot pads. All GM in control lymph nodes were dendritic in shape at the cortical surface and strongly immunoreactive to S-100 protein. In lymph nodes of injected animals, three types of GM were observed at the cortical surface: cells were S-100-protein-positive/phagocytosis-positive, S-100-protein-positive/phagocytosis-negative and S-100-protein-negative/phagocytosis-positive. GM that were S-100-protein-negative/phagocytosis-positive appeared from 1 h onwards, reaching a maximum number at 6 h and then decreasing in number with time at the cortical surface. In the medulla, such cells were observed after 1 h, increasing to a maximal level at 6 h and, thereafter, gradually decreasing in number. These results indicate that expression of S-100 protein in GM is altered by antigen stimulation and that GM move in the lymph nodes. The same pattern of expression of S-100 protein was demonstrated in the FDC 1 week after injection.

CRRP: a guinea pig protein, identified by sequence homology to human CR1, which contains two short consensus repeat motifs and appears not to be transmembrane or secreted.

cDNA from the C4b-binding site of the human C3b/C4b receptor (CR1) was used to find homologous sequences in the guinea pig. This cDNA identified an 18S mRNA species in guinea pig spleen, but not liver. Probing of a guinea pig spleen cDNA library identified clones with identical 1.5-kb inserts, which also hybridized to mRNA in spleen, but not liver. Sequence analysis of the insert revealed a single long open-reading frame coding for a 20,000 Mr protein consisting of two short consensus repeat motifs homologous to human CR1, and unique sequence at the amino- and carboxy-terminals of the short consensus repeats. This sequence did not encode peptides with features of transmembrane domains or signal peptides. Antibody to this complement receptor-related protein-beta galactosidase fusion protein recognized a 20,000 Mr protein in SDS lysates of guinea pig spleen, lymph node, lymphocytes, neutrophils, and peritoneal macrophages. Immunoprecipitation of human serum by this antibody revealed an 180,000 Mr protein reacting both with the anti-guinea pig protein antibody and with anti-human CR1 antibody. Immunoprecipitation of guinea pig serum revealed no protein reacting with the anti-guinea pig protein antibody. Tissue staining of cultured peritoneal macrophages with this antibody showed intracellular staining, as opposed to membrane staining obtained with anti-guinea pig Ig antibody. The lack of membrane expression was confirmed by surface protein radiolabeling experiments and by fluorescent staining of surface proteins. Thus, we have identified a guinea pig protein with homology to human CR1, which may have an unusual property for this class of proteins in that it appears to be intracellular.

Certain lymphoid cells contain the membrane-associated component of the phagocyte-specific NADPH oxidase.

Anionic amphiphiles such as long chain unsaturated fatty acids and SDS were shown to activate the superoxide (O2-) producing NADPH oxidase in a cell-free system derived from sonically disrupted phagocytes (macrophages and granulocytes). O2- production required the cooperation of a membrane associated component sedimenting at 48,000 X g (pi) and a cytosolic factor (sigma). The purpose of the present investigation was to find out whether components pi and sigma were also present in non-phagocytic cells that do not produce O2- when stimulated. It was found that the 48,000 X g pellets of guinea pig lymph node and thymus cell sonicates contained significant amounts of component pi, as shown by their ability to support SDS-elicited NADPH-dependent O2- production when supplemented with macrophage cytosol. Lymph node and thymus pi could be extracted from the membrane by 30 mM octyl glucoside, just as its macrophage-derived equivalent. Combining lymph node and thymus 48,000 X g pellet with autologous cytosol did not yield an active enzyme preparation. Also, cytosol from lymph node and thymus cells could not cooperate with macrophage 48,000 X g pellet, indicating that component sigma was lacking in lymphoid cells. Neither pi nor sigma could be detected in guinea pig kidney, the mouse myeloma cell line MOPC 315 and the canine cell line Cf2Th. The 48,000 X g pellet of all nonphagocytic cells examined contained a b-cytochrome that resembled, by its spectral characteristics, the cytochrome b559 thought to be characteristic of phagocytes. In macrophages, cytochrome b559 represented 80% of b-cytochrome content of the 48,000 X g pellet, whereas in non-phagocytic cells, the equivalent material represented only 50 to 60%. There was no correlation between the presence and quantity of the cytochrome b559-like chromophore in the 48,000 X g pellet of a particular cell type and its ability to cooperate with macrophage cytosol in SDS-elicited O2- production.

Changes in lymphocyte subsets after treatment with cyclophosphamide and during the development of contact sensitivity in the guinea pig

Changes in lymphocyte subpopulations were investigated in guinea pig lymph nodes during the development of contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) and following the injection of cyclophosphamide (CY; 300 mg/kg) using a panel of monoclonal antibodies against guinea pig lymphocyte surface markers. Application of a sensitizing dose of DNFB to the ear resulted in a significant increase in the number of cells recovered from the draining auricular and cervical lymph nodes, 4 and 6 days post sensitization. A significant increase in the number of cells in the contralateral cervical node was found at day 6 but not at day 4. At 4 days postsensitization the proportions of Ia positive lymphocytes were higher than those of immunoglobulin positive B cells in the draining auricular and cervical node and the contralateral cervical lymph nodes suggesting T-cell “activation”. Four and six days after sensitization there were no significant changes in the proportions of Pan T and T suppressor/cytotoxic ( T s c ) positive lymphocytes in the draining auricular and cervical lymph nodes. Although contact sensitivity is “classically” a T-cell mediated phenomenon there was a significant increase in the proportion of B cells found in the draining auricular node 4 days after sensitization as compared to the “normal” auricular node. 1, 2, 3 and 7 days after injection of CY there was a significant depletion in the proportion of B-lymphocytes in the cervical lymph node. This effect was maximal between 2 and 3 days after CY and was paralleled by an increase in the proportion of Pan T positive cells. However, the proportion of T s c remained unchanged, resulting in an increase in the Pan T: T s c ratio, suggesting the presence of a population of CY sensitive T s c cells. Pretreatment with CY 3 days before sensitization with DNFB again resulted in a significant depletion of B-cells 4 days after sensitization in the draining auricular and cervical lymph nodes as compared with animals sensitized to DNFB 4 days previously. Similarly there was a concomitant increase in the proportion of Pan T positive cells, but the proportion of T s c positive cells was unchanged. As this regime of CY results in enhanced contact sensitivity these results are in keeping with a role for B suppressor cells in the guinea pig, although there would also appear to be an additional population of CY sensitive T-suppressor cells which may also play a part in the regulation of contact sensitivity in the guinea pig.

Transfer of autoimmune encephalomyelitis with T lymphocytes in strain 13 guinea pigs

Experimental autoimmune encephalomyelitis (EAE) and/or tuberculin sensitivity were transferred to histocompatible recipients with myelin basic protein-stimulated and/or PPD stimulated guinea pig lymph node T cells previously separated by depletion of B cells ("panning") on rabbit anti-guinea pig Ig antibody-coated Petri plates. The depletion was augmented by complement-mediated lysis using mouse anti-guinea pig B-cell monoclonal antibody (31D2), rabbit anti-mouse Ig, and rabbit complement. B cells did not transfer EAE nor provide protection against active immunization with guinea pig spinal cord antigen.

Accessory cell function of cells isolated from Mycobacterium leprae-induced granulomas

The large cells from Mycobacterium leprae-induced granulomas in guinea pig lymph nodes were separated by Percoll discontinuous density gradient centrifugation and on a fluorescence-activated cell sorter (FACS) using cross-reacting monoclonal antibody to human MHC Class II antigens. Large Percoll-separated cells (83% Class II antigen positive and 52% macrophage-specific antigen positive) and FACS-separated cells are able to act as antigen-presenting cells for T-cell proliferation to PPD. In previous studies, macrophage antigen-positive cells consistently failed to act as accessory cells. This indicates that there is a population of accessory cells which are macrophage antigen negative and MHC Class II antigen positive present in these M. leprae-induced granulomas.

Epithelial and mesenchymal layers of post-term placental and umbilical cord amnion as viewed by SEM

Scanning electron microscopic observations on the luminal aspect of the high-endothelial postcapillary venules (PCV) of lymph nodes of mice, rats, hamsters, and guinea pigs show that the surface topography of these venules differs considerably from that of other venules. Two types of surface relief occur: one in which the endothelial cells bulge deeply into the lumen, thus presenting the appearance of a “cobblestone” pattern and another in which the endothelial cell has plate-like lateral extensions which intertwine with similar extensions of other cells, thus presenting a surface relief of “interlacing plates.” Both patterns occur in the same PCV. The part of the PCV with the interlacing plate pattern is located distally from the section with the cobblestone pattern and continues directly into the smooth-walled larger venules. Lymphocytes enter the wall of the PCV through the cell body of the endothelial cells regardless whether the luminal relief has a cobblestone or an interlacing plate configuration. To a more limited extent, transmural passage of lymphocytes occurs through the walls of larger and smooth-walled venules with low endothelium. The direction of movement of lymphocytes at such sites is not known.

Fibronectin distribution during lymph node development in guinea pig: an immunohistochemical study.

We carried out an immunohistochemical study on mesenteric guinea pig lymph nodes, from the 10th day prepartum till the 26th day postpartum, to assess the role of fibronectin in their organization during development. This glycoprotein is diffusely distributed in embryonic lymph nodes, suggesting a primer function during organogenesis. After birth, in fact, it is less widespread and is mainly localized around sinuses and vessels. Our data, supporting the important role of this glycoprotein during lymph node organization, are in agreement with the results obtained in other tissues and organs.

Effects of non-steroidal anti-inflammatory drugs on lymphocyte activation.

Guinea-pig lymph node lymphocytes were stimulated with mitogen (phytohaemagglutinin) in vitro and lymphocyte activation was measured by tritiated thymidine incorporation (DNA synthesis). Inclusion of non-steroidal anti-inflammatory drugs (NSAIDs) in the culture medium at therapeutic concentrations, frequently exerted an inhibitory effect. Such inhibition could not be attributed to the ability of these drugs to inhibit cyclo-oxygenase or lipoxygenase enzymes. Inhibition by salicylates was not associated with cytotoxic or cytopathic effects, since inhibition was only evident when the drugs were included in the early phase of culture. Other NSAIDs exhibited varying degrees of toxicity, which in some instances may account for observed inhibition. The effects on lymphocyte activation of selective inhibitors of pathways of arachidonic acid metabolism, do not support the proposition that the generation of prostaglandins, thromboxanes, leukotrienes or related compounds is an obligatory step during lymphocyte activation.

Production and characterization of antisera against guinea pig macrophage activating factor.

Guinea pig lymph node cells stimulated with concanavalin A (Con-A) were used as a source of lymphokines. Purifications by molecular sizing columns yielded sufficient quantities of macrophage activating factor (MAF)-active fractions to allow immunization of rabbits. An antiserum was produced which is capable of specifically inhibiting MAF activity, as well as blocking the activity of macrophage migration inhibiting factor (MIF), but not of skin reactive factor. Antisera raised against control supernatants derived from lymph node cells not stimulated with Con-A, failed to specifically inhibit any of these lymphokines. Additionally, anti-MAF but not anti-control antibodies suppressed completely contact sensitivity to dinitrochlorobenzene.

Ultrastructural localization of 2,4-dinitrophenyl groups on draining lymph node cells of guinea pigs following skin painting with 2,4-dinitrochlorobenzene: I. Scanning immunoelectron microscopic studies.

ABSTRACTThe distribution of DNP groups on cells taken from draining lymph nodes of guinea pigs 12 hours after painting the skin with DNCB was examined by scanning immunoelectron microscopy using antibody directed against DNP groups with bacteriophage T4 as a visual marker. The studies showed that DNP groups were distributed on the surface of the cells, demonstrating some differences in distribution pattern among the cells. The incidence of cells on which DNP groups were detected was approximately 60% in the normal animals, higher than that detected by a previous immunofluorescent study. The incidence was markedly reduced to approximately 3% by pretreatment with tolerogen, DNBSO3Na. DNP groups were detected on the surface of the cells forming both rabbit red cell rosettes and complement coated red cell rosettes. It is suggested that DNCB penetrates through the skin into the lymphatics carrying lymph from the painted site and combines with cell membrane components in the peripheral lymphoid system.