Influence of heparin and protamine on enlargement of the draining lymph node during induction of EAE

Popliteal lymph node enlargement four days after immunization with encephalitogenic guinea pig basic protein in Freund's complete adjuvant (EGPBP-FCA) was less in heparin treated and greater in protamine-treated Lewis rats than in salineinjected controls. These agents were without influence on the node enlargement occasioned by Freund's adjuvant alone or on node size in nonimmunized rats. Decreased cell trapping in the node in heparinized rats and increased trapping in protamine-treated rats immunized with EGPBP-FCA is suggested.     

Tracking of V beta 8.2-positive encephalitogenic T cells by complementarity-determining region 3 spectratyping and subsequent Southern blot hybridization in Lewis rats after neuroantigen sensitization

Pathogenic T cells in organ-specific autoimmune diseases use a limited number of TCR alpha- and beta-chains. In experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats by immunization with myelin basic protein, encephalitogenic T cells mainly use Vbeta8.2 TCR and clonal expansion of the Vbeta8.2 spectratype containing the EAE-specific complementarity-determining region 3 (CDR3) sequence, DSSYEQYFGPG, is found in the spinal cord throughout the course of clinical EAE. In the present study we performed temporal and spatial analyses of Vbeta8.2 spectratype expansion by CDR3 spectratyping and subsequent DNA hybridization with a probe specific for the encephalitogenic CDR3 sequence to elucidate the kinetics of encephalitogenic T cells during the induction phase after neuroantigen sensitization. It was demonstrated that Vbeta8.2 spectratype expansion and/or the positive signal in Southern blot were first detected in the regional lymph nodes as early as day 3 postimmunization and was disseminated over the lymphoid organs by day 6. Because perfusion of immunized rats with PBS erased the positive signals on day 3 postimmunization, the majority of Vbeta8.2-positive encephalitogenic T cells at the very early stage would reside within the lymphatic or blood vessels. Furthermore, removal of the draining lymph node 1, 3, and 6 days after immunization in the foot pad did not ameliorate clinical EAE. These findings strongly suggest that encephalitogenic T cells disseminate throughout the whole body very rapidly after sensitization. Analysis of pathogenic T cells at the clonal level provides useful information for designing effective immunotherapy.     

Metallic tin-induced lymphadenopathy in rat strains and hybrids

Metallic tin powder, injected into Lewis rats obtained from three different sources, caused enlargement of the regional draining lymph nodes. The histopathology featured epithelioid cell granulomas around phagocytosed particles of tin and an intense hyperplasia of plasma cells. The same material injected into August rats enlarged the lymph nodes but the enlargement was caused by granulomas without a major concomitant plasma cell response. In most other strains, tin produced less lymph node enlargement and the plasma cell response was minimal. However, F1 hybrids of Lewis rats with either the August, Brown-Norway (BN), or Dark Agouti (DA) strains developed plasma cell hyperplasia similar to that seen in the parental Lewis strain. The response to tin was the same whether the tin was injected into the feet or into the peritoneal cavity. Thus, the lymph node response to metallic tin varied from a slight, banal response to insoluble foreign particles, to an exuberant granulomatous hyperplasia, to an intense plasmacellular hyperplasia, depending on the genetic characteristics of the subjects.     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

Experimental allergic encephalomyelitis and cellular immunity in the Lewis rat

The encephalitogenic difference between purified guinea pig and bovine myelin proteins in the Lewis rat is reflected by the two molecules' lack of crossreactivity in the migration inhibition test. Peritoneal exudate cells from rats injected with guinea pig or bovine derived myelin basic protein in Freund's complete adjuvant demonstrate substantial migration inhibition to the sensitizing antigen but little inhibition when cultured in the presence of the other basic protein. The cellular reactivity to guinea pig basic protein is present throughout the induction phase of Experimental Allergic Encephalomyelitis and persists after the recovery of the rats from the paralytic state. Substantial cellular reactivity is also demonstrated to bovine basic protein even though this molecule shows minimal encephalitogenic activity in the Lewis rat. Minimal lymphocyte transformation could be demonstrated to either of the basic proteins, although the immune cells react strongly to the plant mitogen phytohemagglutinin and to a Mycobacterium tuberculosis antigen.     

3H-deoxythymidine incorporation in graft-versus-host disease in the Norway rat. I. Liquid scintillation studies.

Graft-versus-host-disease was produced in newborn Brown Norway (BN) rats with an intravenous (iv) injection of adult allogeneic Lewis (L) lymph node cells (experimental) and the response was compared to littermates injected with adult syngeneic BN cells (control). By 4 days the reaction in the spleen of experimental animals was such that the spleen index was 1.70 and 2.58 on day 7, and continued to increase until death. A one hour iv pulse of tritiated deoxythymidine (3HdT) administered to experimental and control animals revealed a whole organ peak incorporation of 3HdT on day 6 in experimental spleens. A second larger peak occurred on day 10 in the experimental spleen as compared to a single peak at days 6 or 7, respectively, in the experimental mesenteric and combined superficial lymph nodes. However, analysis of the incorporation of 3HdT with respect to organ weight revealed a peak incorporation in animals receiving L cells on day 4--6 with a second smaller peak on day 10 in the experimental spleen and again a single peak on day 5 or 6 in the lymph nodes. Total 3HdT incorporation within both experimental lymph node compartments became less than controls by day 15 even though experimental nodes had a larger mass. 3HdT incorporation per milligram tissue weight decreased in all tissue compartments of experimental animals by day 13--14. The contribution of donor and host cell proliferation to the various peaks observed is discussed.     

Trypanosoma equiperdum: Movement from the dermis

Twelve dogs were injected intradermally with 352,770 to 14,391,660 Trypanosoma equiperdum and afferent and efferent lymph, lymph nodes, and blood examined by mouse inoculation at minute, hourly, and daily intervals following inoculation. The log dosage of trypanosomes given each dog was closely related to their body weight (P < 0.01). Afferent lymph contained trypanosomes as soon as 5 and 27 min after inoculation. Lymph nodes on the side of injection became positive within 5 min of injection, while those on the contralateral side remained negative for at least 120 min after injection. Blood contained trypanosomes as soon as 5 min after injection, although the average time for all dogs, before trypanosomes were demonstrated in the blood, was 40 min postinjection. Efferent lymph did not contain organisms until 25–76 hr after inoculation. We consider this sequence to indicate that T. equiperdum can leave the dermis in afferent lymphatics, reach the local lymph node, invade the blood stream from this site, and only after a day or longer do they leave the node via the efferent lymphatics.     

Peptides of myelin basic protein are encephalitogenic in rats without the aid of emulsions.

Immunization with peptides is usually done with the aid of Freund's adjuvant. Using peptides derived from myelin basic protein, we show that aqueous solutions can be antigenic (encephalitogenic in this instance) in Lewis rats. The first procedure involved multiple doses of aqueous peptide, increased absorption into the lymphatic system from the peritoneal cavity in the postinflammatory state, and the use of pertussis vaccine. Three different peptides containing the major encephalitogenic site were active in this system, with the activity somewhat proportional to the size of the fragment. The second procedure, the direct delivery of peptide to lymph nodes by percutaneous inoculation, was equally successful and did not require the use of pertussis vaccine.     

Neutral protease activity in lymphocytes of lewis rats with acute experimental allergic encephalomyelitis

Lymphocytes from popliteal and inguinal lymph nodes of Lewis rats with acute EAE as a result of injection of lyophilized guinea pig myelin in Freund's complete adjuvant exerted strong proteolytic activity at neutral pH toward myelin basic protein. After injection of myelin the level of proteolytic activity remained about the same as that in lymphocytes from Freund's adjuvant-injected controls until about day 10 after injection, just before the onset of paralytic symptoms; then the proteolytic activity increased to approximately double its former level. Myelin basic protein was hydrolyzed by whole lymphocytes, but more activity was unmasked by homogenization. Similar results were also obtained using lymphocytes from thymus of EAE and control animals. Lymphocytes with high levels of proteolytic activity were not absorbed by glass wool, did not stain with neutral red, nor did they phagocytose antibody-coated sheep red blood cells. Thymus and lymph node lymphocytes cleaved myelin basic protein to three major peptides and a fourth minor peptide, while spleen lymphocytes hydrolyzed basic protein at only one point resulting in two peptides whose molecular weights added up to that of myelin basic protein. The protease activity was inhibited by 5×10^–3 Mp-chloromercuribenzoate and by phenylmethyl sulfonyl fluoride, TPCK, and soybean trypsin inhibitor, therefore the enzymatic activity probably depends on a serine residue and a sulfhydryl group. The bulk of the enzymatic activity is mostly membrane bound with the highest specific activity and total activity contained in a lysosomal-mitochondrial fraction. In view of the infiltration of lymphocytes into the brain substance in acute EAE, it is suggested that these cells may contribute to the destruction of myelin which is usually attributed to the monocyte or macrophage.     

Malnutrition interferes with the unique lymphadenopathy induced in lewis rats by metallic tin

Metallic tin powder injected into Lewis rats caused marked enlargement of draining lymph nodes with prominent hyperplasia of plasma cells. A low level of dietary protein (8%) or deficiency of choline interfered with the cellular proliferation and reduced the size of the nodes by nearly onethird compared to a normal protein diet (20%). A high level of protein in the diet (50%) did not augment the effect of metallic tin powder beyond that achieved by a level usually considered adequate (20%).     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Modulation of adoptive cell transfers in experimental allergic encephalomyelitis by antigen treatment of donors

Experimental allergic encephalomyelitis has been adoptively transferred using lymph node cells from Strain 13 guinea pig donors sensitized with purified encephalitogenic myelin basic protein. Adoptive cell transfer was used to examine the immunocompetence of lymph node cells obtained from guinea pigs protected from disease development by treatment with MBP. Lymph node cells from guinea pigs unresponsive to EAE challenge do not adoptively transfer disease. Cells obtained from guinea pigs treated with MBP following encephalitogenic challenge are competent in adoptive transfer with respect to pathologic lesions, but not clinical disease. The clinical and pathologic responses of recipients of the histocompatible lymphocyte populations are similar to those seen in the treatment-matched donor controls, suggesting that under these circumstances lymphoid cells, rather than circulating soluble factors, are responsible for disease induction and suppression.     

A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.     

The passive transfer of protective immunity against Angiostrongylus cantonensis with immune lymph node cells from different lymphoid tissues in rats

-Yong W. K. and Dobson C. 1982. The passive transfer of proctective immunity against Angiostrongylus cantonensis with immune lymph node cells from different lymphoid tissues in rats. International Journal for Parasitology12: 423–425. Lymph node cells from the posterior cervical and mesenteric lymph nodes of immune rats passively protected syngeneic recipient rats against Angiostrongylus cantonensis better than cells from the spleen, thymic and inguinal lymph nodes either as reduced worms burdens and/or stunted growth. No antibody was detected in the sera of recipient rats after transfer of the cells and before infection which suggested that the protection was cell- rather than antibody-mediated.     

PROTEOLYTIC ENZYMES AND EXPERIMENTAL DEMYELINATION IN THE RAT AND MONKEY

Abstract— Visible lesions from monkeys with acute experimental allergic encephalomyelitis (EAE) induced by injection of purified myelin basic protein were assayed for acid proteinase, for a neutral proteinase at pH 6·5, and one lesion was measured for cathepsin A. Acid proteinase was increased to 152–176 per cent of levels in normal-appearing brain areas, neutral proteinase increased to 220–258 per cent, and the one lesion assayed for cathepsin A was 840 per cent of control. These enzymes were measured in the brain stem of Lewis rats with acute EAE as a result of basic protein injection and compared to Freund's adjuvant-injected controls. Acid proteinase was increased significantly to an average level of 128 per cent of control, the increase in neutral proteinase was not significant, and cathepsin A levels were 258 per cent of control, a highly significant increase. The rise in cathepsin A levels was not seen until the onset of paralytic symptoms. The brain stem of Wistar rats treated with whole spinal cord which show EAE in a milder form than the Lewis rat did not contain significantly higher enzyme levels than the control. The increases in acid proteinase and cathepsin A in brain stems were compared to levels of these enzymes in lymph nodes of EAE, Freund's adjuvant-injected controls and uninjected controls. The level of acid proteinase of lymph nodes/g protein did not change appreciably in the course of EAE development in the Lewis and Wistar rats and was about 3–4 times the activity in the brain stem. The cathepsin A in the inguinal lymph nodes of Wistar and Lewis rats injected with whole spinal cord in Freund's adjuvant increases to a level 2× that of the lymph nodes of the uninjected control. The cathepsin A levels in these activated lymph nodes was 6–8 × that of the control brain stem. The lymph nodes of Lewis and Wistar rats injected with Freund's adjuvant alone showed the same increase in cathepsin A as those from rats injected with spinal cord. The brain stem of rats undergoing severe demyelination as a result of chronic administration of triethyl tin did not show the enzyme increases. These results are compatible with the theory that proteolytic enzyme increases in EAE (and probably multiple sclerosis) are due to the invasion of mononuclear cells, some of which are probably lymphocytes. Whether or not these enzymes participate in the actual dissolution of myelin is unknown.     

Antigen recognition in autoimmune encephalomyelitis and the potential for peptide-mediated immunotherapy

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in (PL/J x SJL)F1 mice. Amino acids that determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides that bind MHC yet do not cross-react with the autoantigen. A peptide analog of the encephalitogenic epitope is shown to be "heteroclitic" for MHC binding and activation of encephalitogenic T cells in vitro. This analog is not immunogenic for encephalitogenic T cells in vivo and is shown to inhibit disease that is induced by the autoantigen itself.     

Reutilization by maternal and embryonal cells of nuclear materials from H3-thymidine labeled lymphocytes introduced into the lumen of intestine of rats

Summary H³-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.     

The role of dendritic cells in the initiation of immune responses to contact sensitizers. I. In vivo exposure to antigen

Twenty-four hours after skin painting mice with picryl chloride (PIC) there was a four- to fivefold increase in the numbers of dendritic cells (DC) isolated from the lymph nodes. These DC initiated primary proliferative and cytotoxic responses when added to cultures of normal syngeneic lymph node cells. The proliferative response was enhanced when the donors of the responding lymph node cells were sensitized with the same antigen. Contact sensitivity developed in syngeneic mice injected into the footpads with 30,000-50,000 DC from lymph nodes of mice painted with picryl chloride 1 day previously. Thus, 1 day after skin painting mice, there were dendritic cells in the draining lymph nodes which were able both to initiate primary stimulation of lymphocytes in vitro and to sensitize recipient mice to give specific delayed hypersensitivity reactions.     

Epithelioid cell granuloma induction in the guinea pig by haptenated Mycobacterium leprae

Fluorescein isothiocyanate (FITC)-conjugated Mycobacterium leprae (FITC-M. leprae) was injected intradermally into the ears of guinea pigs and granuloma formation in the draining postauricular lymph nodes was studied. At 2 weeks, there was an increase in weights and infiltration of the draining lymph nodes in half of the animals injected with FITC-M. leprae. At 5 weeks, there was a significant increase in the weights and infiltration of these draining lymph nodes in the guinea pigs injected with haptenated M. leprae compared with those injected with unconjugated M. leprae. At 5 weeks, there was also a significant increase in delayed type hypersensitivity responses to 25 μg purified protein derivative. Histologically, epithelioid cell granulomas were seen in these lymph nodes as early as 2 weeks when FITC-M. leprae was used as the source of antigen. Enhancement in the immunogenicity of M. leprae by conjugation with FITC has been postulated.     

Preoperative lymphoscintigraphy guided sentinel lymph node biopsy in malignant melanoma

The authors present preliminary experience with preoperative sentinel lymph node biopsy carried out with lymphoscintigraphy in patients with malignant melanoma. PATIENTS AND METHODS: In the present study patients operated for primary cutaneous malignant melanoma of moderate and high severity were included. On the day of surgery isotope labelled colloid was injected intradermally around the tumor to indicate the lymphatics and to obtain basic information about the localization of the sentinel lymph node(s).During surgery the lymph node(s) previously visualized by the injection of patent-blue staining were detected with the aid of a gamma probe. Simultaneously, the excision of the primary tumor was extended. Histologically verified metastasis in the surgically removed lymph node(s) necessitated block dissection possibly within two weeks. RESULTS: The distribution of patients (19) according to tumor localisation: 2 - upper extremities; 9 - lower extremities; 2 - sacral region; 6 - trunk. Tumor thickness ranged from <1.5 mm (6 patients) to 1.5-3 mm (5 patients) and to >3 mm (8 patients). In two cases the identification of the lymph node has failed. Positive sentinel ymph nodes were detected in two patients. It is noteworthy that with one patient the sentinel lymph node was not regional but intransit. This study was aimed at the development of a suitable method. Further on we wish to try it in prospective randomized studies.