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PubMed2001

Germinal center reaction in the joints of mice with collagen-induced arthritis: an animal model of lymphocyte activation and differentiation in arthritis joints.

OBJECTIVE: To establish an animal model and provide a basis for investigating the role of germinal center (GC) reaction in autoimmune arthritis. METHODS: DBA/1 mice were immunized with bovine type II collagen (CII) to elicit collagen-induced arthritis (CIA). Sections of arthritic joints were examined by in situ immunohistochemical studies, and purified cells from affected joints were subjected to flow cytometry analysis. RESULTS: De novo GC reaction was induced in the arthritic joints of male DBA/1 mice by immunization with bovine CII. In comparison with GCs formed in lymphoid tissues, such as spleen and lymph nodes, we found that these GCs formed in the joint tissues of mice with CIA were morphologically typical, as determined by immunohistologic and flow cytometric assays. CONCLUSION: The local immune responses in murine CIA induced ectopic GC formation, as observed in the synovial tissues of patients with rheumatoid arthritis. This system will allow for the first time the direct study of the role of the GC reaction in autoimmune arthritis in an animal model.

Animals↗
PubMed2003

Bacterial translocation up-regulates GTP-cyclohydrolase I in mesenteric vasculature of cirrhotic rats.

In cirrhosis, arterial vasodilation and the associated hemodynamic disturbances are most prominent in the mesenteric circulation, and its severity has been linked to bacterial translocation (BT) and endotoxemia. Synthesis of nitric oxide (NO), the main vasodilator implicated, is dependent on the essential cofactor tetrahydrobiopterin (BH(4)). The key enzyme involved in BH(4) synthesis is GTP-cyclohydrolase I (GTPCH-I), which is stimulated by endotoxin. Therefore, we investigated GTPCH-I activity and BH(4) biosynthesis in the mesenteric vasculature of cirrhotic rats with ascites, as well as their relationship with BT and endotoxemia, serum NO, and mean arterial pressure (MAP). GTPCH-I activity and BH(4) content in mesenteric vasculature was determined by high-performance liquid chromatography. BT was assessed by standard bacteriologic culture of mesenteric lymph nodes (MLNs). Serum endotoxin was measured by a kinetic turbidimetric limulus amebocyte lysate assay, and serum NO metabolite (NOx) concentrations were assessed by chemiluminescence. BT was associated with local lymphatic and systemic appearance of endotoxin and was accompanied by increases in serum NOx levels. GTPCH-I activity and BH(4) content in mesenteric vasculature were both increased in animals with BT and correlated significantly (r = 0.69, P <.01). Both GTPCH-I activity and BH(4) levels significantly correlated with serum endotoxin and NOx levels (r = 0.69 and 0.54, 0.81 and 0.53, P <.05). MAP (a marker of systemic vasodilatation) correlated with endotoxemia (r = 0.58, P <.03) and with GTPCH-I activity (r = 0.69, P <.01). In conclusion, in cirrhotic animals BT appears to lead to endotoxemia, stimulation of GTPCH-I, increased BH(4) synthesis, and further enhancement of vascular NO production that leads to aggravation of vasodilatation.

Animals↗
PubMed1981

Quantitative oestradiol receptor values in primary breast cancer and response of metastases to endocrine therapy.

Quantitative values if oestradiol receptor were determined in the primary breast cancer of 526 patients, 106 of whom have so far required hormonal therapy for metastatic disease (premenopausal patients-oophorectomy; postmenopausal patients-tamoxifen 20 mg twice daily). The rate of response to treatment was significantly higher in patients with receptor-positive primary cancers, and the likelihood of response further increased in proportion to the measured receptor concentration. Preliminary data also suggest that patients with high receptor values in the primary tumour will enjoy a longer remission than those with low measured values. Assays of oestradiol receptor were also carried out on biopsy specimens of accessible metastases, largely skin and lymph-node recurrences, in 24 patients who were treated mainly with local measures. Receptor status was the same in both the primary and the secondary tumour in only 18 patients.

Adult↗
PubMed1977

In vivo and in vitro effects of acute graft-versus-host serum in the rat.

Pooled serum from 6- to 8-week-old female Lewis x Brown Norwegian F1 hybrid (LBNF1) rats undergoing acute local graft-versus-host (GVH) reactions was found to have the ability, when compared with normal F1 serum, to increase node weight in a popliteal lymph node weight gain assay in syngeneic animals. Suspension of donor cells in normal F1 serum on one side and corresponding concentrations of GVH serum on the contralateral side resulted in an average increase in popliteal node weight of 28.8 per cent on the side receiving GVH serum. Of the 64 animals tested, 50 had larger nodes on the GVH serum side. This effect was not impaired by heating (56 C, 30 min) or by rapid freezing (-75 C) and thawing of the serum. Preinjection of the serum into the footpads 2 hr before donor cells also gave increased node weights as did injection of the serum i.p. In contrast, GVH serum was found to inhibit unidirectional mixed lymphocyte cultures of seven different allogeneic rat strain combinations, the inhibition ranging from 35 to 74 per cent. In addition, the blastogenic cell responses of three different strains to phytohemagglutinin P were inhibited between 45 and 77 per cent. The relationship between these two phenomena, in vivo increase in node weight and in vitro inhibition of blastogenesis, is not clear and is the object of further investigation.

Animals↗
PubMed1983

IgA antibodies in the bile of rats. III. The role of intrathoracic lymph nodes and the migration pattern of their blast cells.

Eight weeks after rats had had their mesenteric lymph nodes (MLN) removed surgically, they were found to be still able to generate substantial titres of biliary IgA-antibodies after antigens were injected into their Peyer's patches. This suggested that systemically significant IgA production could be induced in extra-abdominal lymphoid tissue. It was found that the intrathoracic lymph nodes (ITLN) were an important source of IgA production. These nodes could be stimulated to produce biliary antibody by introducing antigen either into the peritoneal cavity or directly into the thorax. Cells forming IgA were identified in the ITLNs by haemolytic plaque assays and immunoperoxidase techniques. In spite of this, immunoblasts obtained from the ITLNs, and labelled with 125IUdR did not localize in the gut after i.v. injection to anywhere near the extent that immunoblasts from the MLN did. Instead they seemed to have a predilection for localizing in the lungs.

Animals↗
PubMed1987

The usefulness of CEA as an indicator for early detection and a guide to the treatment of recurrent gastric cancer.

The usefulness of carcinoembryonic antigen (CEA) as an indicator for recurrence and a guide to the treatment was evaluated from a retrospective analysis of 88 patients with recurrent gastric cancer. Sixty-two of these patients (70.5 per cent), 25 of whom had a preoperative positive assay, and 37 a negative assay, had elevated levels of CEA after disease progression. Averaged CEA level in patients with liver metastasis was significantly higher (872 ng/ml) than in those with peritoneal metastasis (68 ng/ml), with lymph node metastasis (103 ng/ml) or with local metastasis (93 ng/ml) (p less than 0.01). An elevation of CEA was found prior to the clinical manifestation of recurrence, and the average lead time was 4 months. In 25 patients with a lead time of more than 4 months, survival time after CEA elevation was 13.3 months, which was longer than the 6.5 months of 28 patients with less than 4 months. Thirty-seven of the 88 patients were treated after recurrence. The average survival period after the detection of recurrence was 9.4 months in patients with surgical treatments followed by chemotherapy, 5.9 months in those with chemotherapy alone and 3.8 months in those with surgery alone. The average survival period of 26 patients with positive CEA assays in recurrence was 5.1 months longer than of patients with negative assays. This fact suggested that early detection of recurrence followed by various treatments, in the elevated CEA group, contributes to favorable results.

Adult↗
PubMed1977

Effects of trypan blue treatment on the immune responses of mice.

It has been reported that trypan blue treatment decreases the nonspecific resistance of mice to transplanted tumors and inhibits the in vitro cytotoxic activity of activated macrophages. We wished to determine whether this effect of trypan blue could be due to a selective inhibition of certain macrophage functions or whether it reflected a broader form of immunosuppression. We therefore tested the effects of trypan blue on a variety of immunological responses. Treatment of mice with trypan blue delayed their rejection of skin allografts and transplants of a highly antigenic syngeneic ultraviolet light-induced tumor. Trypan blue treatment of either donor or recipient decreased the local graft-versus-host reaction. Filtration of lymph node cells from trypan blue-treated donors on a nylon wool column before use in the graft-versus-host assay abrogated the depressive effect of trypan blue. A transient reduction in the blastogenic response of spleen cells to concanavalin A and lipopolysaccharide mitogens was observed after a single injection of trypan blue, but the response of lymph node cells was unaffected. The depressed response of splenic lymphocytes was not entirely reversed by removal of adherent cells. The primary and secondary hemagglutinin responses to sheep erythrocytes were unaffected in trypan blue-treated mice, and the proportion and phagocytic activity of thioglycolate-induced peritoneal macrophages were also unaltered. We conclude that treatment of mice with trypan blue selectively inhibits certain macrophage functions but, at high doses, it can also inhibit some lymphocyte activities.

Agglutinins↗
PubMed1983

The immune status of the rat with carcinoma of the bowel.

Studies were made to determine whether rats receiving 1,2-dimethylhydrazine (DMH) to induce bowel cancer were immunologically compromised during tumour development. General cellular immunodepression was observed in DMH rats in nonspecific assays of T-cell and killer-cell (K-cell) function and later in allograft response and in vitro tumour immunity tests. B-cell levels in local lymph nodes increased significantly very late in tumour growth. It is suggested that DMH itself was exerting immunosuppressive as well as carcinogenic effects while it was administered. After withdrawal of DMH some immune faculties (tumour immunity, B-cell response) reappeared, so it is unlikely that the immune suppression observed was a result of factors produced by the developing neoplasm. There are indications that DMH administration in the rat is immunosuppressive and that tumour growth may be facilitated by this action and not by tumour reactive lymphocytic depletion or paralysis associated with blocking factors or suppressor cell production.

1,2-Dimethylhydrazine↗
PubMed2001

Subcellular localization of the autoimmune regulator protein. characterization of nuclear targeting and transcriptional activation domain.

The autoimmune regulator (AIRE) gene, defective in the hereditary autoimmune disease APECED, encodes a transcriptional regulator protein. AIRE is expressed in the medullary epithelial cells and monocyte-dendritic cells of the thymus with lower expression in the spleen, fetal liver, and lymph nodes. At the cellular level, AIRE is located in microtubular structures of the cytoskeleton and in discrete nuclear dots resembling ND10 nuclear bodies. We studied the determinants of the targeting of AIRE into these structures. We report here that the N-terminal HSR domain confers localization to the microtubular network whereas the C-terminal region contains a second nuclear localization signal. We also demonstrate that the consensus nuclear localization signal of AIRE is functional and that the HSR domain harbors a nuclear export signal. Accordingly, the nuclear export inhibitor leptomycin B partially inhibits the nuclear export of AIRE. From a functional standpoint, we show that AIRE can activate the interferon beta minimal promoter in a transfection assay and demonstrate that the transcriptional activating function of AIRE is mediated by its two plant homeodomain (PHD) zinc fingers.

Animals↗
PubMed1994

Morphology of popliteal lymph node responses in Brown-Norway rats.

The popliteal lymph node (PLN) assay has been proposed as a tool to predict in rodents those xenobiotics likely to induce autoimmune reactions in humans. To further validate this assay and to study the mechanisms involved, histologic changes in PLNs from rats injected with streptozotocin, diphenylhydantoin, pure acetone, or 50% ethanol were compared to a local graft-versus-host (GvH) reaction. This study suggests that routine histology of PLNs is instrumental to discard primary irritants. In addition, the hypothesis of a GvH-like mechanism in positive PLN responses is supported by the finding that the reference compounds streptozotocin and diphenylhydantoin produced histologic changes similar to a "true" local GvH reaction.

Acetone↗
PubMed1978

Successful cardiac allografts in syngeneic radiation chimeras.

Successful cardiac allografts were accomplished across the major histocompatibility complex of rats. LEW and F344 (Ag-B2) rats were lethally irradiated and grafted with WF (Ag-B1) hearts on day 0. Either on day 0 or day 2, the hosts were repopulated with syngeneic hemopoietic cells. The best results were obtained (86%) when a mixture of 3.0 x10(7) non-adherent syngeneic bone marrow and thymus cells were used to repopulate the recipients. In contrast, all of the WF to LEW heart grafts were rejected within 30 days if syngeneic thoracic duct and bone marrow cells were used to repopulate the host. Tolerant rats bearing a functioning WF heart graft were able to mount a normal antibody response to SRBC and a proliferative response to Con A. They accepted a second WF heart or a WF kidney graft but rejected WF skin and bone marrow grafts as well as "third-party" ACl or BN hearts. The lymphocytes of tolerant rats had a reduced response to WF antigens as assayed by local or systemic graft-versus-host reactions in (LEW x WF)F1 recipients. Tolerance to the WF hearts was resistant to a large innoculum of normal spleen and lymph node cells. The unresponsive state could be transferred to unirradiated LEW rats with a mixture of spleen, thymus, lymph node and bone marrow cells.

Animals↗
PubMed1987

Immune response against P815X2 mastocytoma growing in syngeneic DBA/2 mice. IV. Lymph node immunoreactivity and cell-mediated cytotoxicity.

The in vitro cytotoxicity assay and the morphological in vivo analysis were used to follow the T cell reaction in tumor draining lymph nodes from DBA/2 mice bearing subcutaneously developing P815X2 mastocytoma. Mononuclear cells separated from local lymph nodes between days 8 and 14 following tumor inoculation were able to kill P815X2 cells in vitro. The maximal tumor-specific cytotoxicity was measured on day 10. Changes in lymph node morphology and in cellular contents of individual lymph node compartments were seen from day 12 onwards. The extensive expansion of the paracortex and the enlargement of the whole lymph node reached the peak on day 14. On days 12 and 14, a slight increase was also observed in the proportion of T lymphocytes. The results indicate that the tumor-specific cytotoxicity was detectable in tumor draining lymph nodes four days earlier than the morphological and cellular changes. The maximal cytotoxicity preceded the morphological peak reactions by two to four days.

Animals↗
PubMed1990

Mapping of the rat mast cell granule proteinases RMCPI and II by enzyme-linked immunosorbent assay and paired immunofluorescence.

The distribution of the rat mast cell granule proteinases, rat mast cell proteinase I and II (RMCPI and II respectively) has been determined in rat tissues with the aid of highly sensitive and specific enzyme-linked immunosorbent assays (ELISA) and paired immunofluorescence. The major source of RMCPII is the gastrointestinal tract, although low concentrations were also detected in non-mucosal sites including thymus, mesenteric lymph nodes, liver, bone marrow, heart, kidney and spleen. Cellular localization by paired immunofluorescence showed that most cells contained either RMCPI or RMCPII, although a minor subpopulation in which individual cells contained both proteinases was also identified in a few tissues. RMCPII-containing cells predominated at mucosal surfaces but were also found in non-mucosal tissues. Individual cells expressing both RMCPI and II were present in lung, liver mesenteric lymph node and submucosa of stomach and were occasionally represented amongst serosal cells from the peritoneal cavity. Connective tissue mast cells of skin and tongue were identified as major sources of RMCPI, although this proteinase was widely distributed in all tissues examined. The present study demonstrates the heterogeneity of mast cell proteinase phenotypes in the rat and emphasises the difficulties in determining mast cell subtypes on tissue location alone.

Animals↗
PubMed1983

Mechanism of the A/Ph.MC.S1 tumor graft rejection in syngeneic mice.

A considerable number of normal mice injected with a low dose of A/Ph.MC.S1 syngeneic methylcholanthrene-induced tumor cells showed regression of the tumor after a transient period of growth. The regressor mice eliminated a challenge inoculum in an accelerated fashion. Splenic lymphocytes from such regressor mice inhibited the growth of the same tumor in the local adoptive transfer assay. This capacity required the presence of thymus-derived cells. The regressor animals developed a delayed-type hypersensitivity response against the tumor In vivo and their lymph-node cells produced macrophage migration inhibitory factor in the presence of tumor cells in vitro. The growth of this tumor was facilitated by treating the primary recipients with carrageenan, and the strong tumor-inhibitory capacity of regressors was also depressed by this agent. Early macrophage infiltration of the tumor in regressor mice was demonstrable histochemically, preceding the inhibition of tumor growth. A lymphokine-producing T cell-mediated, macrophage-dependent delayed-type hypersensitivity-like mechanism is proposed to be the dominant mechanism of the resistance in this tumor-host model.

Animals↗
PubMed1984

Persistence of an antigen recognized by Crohn's disease sera during in vivo passage of a Crohn's disease-induced lymphoma in athymic nude mice.

Previous studies from this laboratory have shown that lymphoma and lymph node hyperplasia develop in athymic nude mice injected with Crohn's disease tissue filtrates. In addition, use of an indirect immunofluorescence assay has demonstrated that an antigen(s) in these lymphoid tissues is recognized by sera from patients with Crohn's disease. In our study, one such lymphoma was passed through 10 generations of athymic nude mice and used for immunofluorescence studies. Suspended cells (1 to 5 X 10(8)/mouse) from the primary lymphoma and from the lymphomas that developed in mice of each subsequent generation were injected subcutaneously into groups of three to eight mice. As a control, lymphoma induced in a nude mouse by the injection of sarcoid lymph node filtrate was passed through successive generations in the same manner as the Crohn's disease-induced lymphoma. Lymphoma developed locally in 81% of recipient mice 3 to 6 weeks after injection of suspended cells. In 47 mice, representing 10 generations, Crohn's disease-related lymphoma developed at the sites of injection and at axillary or inguinal lymph nodes. These tumors were examined by immunofluorescence assay using a panel of 20 sera coded for Crohn's disease or control. Immunofluorescence assay yielded positive results in 61% of the lymphomas and 59% of the lymph node with sera from patients with Crohn's disease, but not with control sera. Control lymphomas did not stain with any serum. Our studies demonstrate the transmissibility or inducibility of an antigen(s) in lymphoid tissue of athymic nude mice that is recognized by sera from patients with Crohn's disease, but not by control sera.

Adult↗
PubMed1986

Intradermal administration of 4-hydroperoxy-cyclophosphamide during contact sensitization potentiates effector T cell responsiveness in draining lymph nodes.

4-Hydroperoxy-cyclophosphamide (4-HPCY) is an in vitro active form of cyclophosphamide. In a previous study, using an in vivo contact sensitivity model in the guinea pig, we demonstrated that intradermal injection of small amounts (50-200 micrograms) of 4-HPCY at the sensitization site resulted in strong potentiation of contact hypersensitivity (Boerrigter and Scheper, 1984). It was postulated that 4-HPCY induces a local decrease of feedback control within the draining antigenically stimulated lymph nodes. The present data are in support of this view: Lymph node hyperplasia induced by contact sensitization (to dinitrochlorobenzene or oxazolone) was further enhanced by 4-HPCY treatment. The paracortical area was preferentially enlarged. 4-HPCY-treated lymph nodes showed an augmentation of hapten-specific T effector cell function as determined in transfer experiments. The response of such lymph node-derived cells to the T cell mitogen PHA was enhanced. Although 4-HPCY treatment resulted simultaneously in a decrease in responsiveness of draining lymph node-derived cells to the B cell mitogen lipopolysaccharide, anti-hapten antibody production was not affected. The present study demonstrates that important similarities exist between the effects of local 4-HPCY treatment and systemic cyclophosphamide pretreatment on the immune response. As systemic treatment with a high dose of cyclophosphamide is known to have serious side effects, the present local protocol provides a new attractive and versatile strategy for T cell immunopotentiation.

Animals↗
PubMed1983

Differences in in vivo distribution and homing of T cell subsets to mucosal vs nonmucosal lymphoid organs.

The migratory properties of Lyt-2- and Lyt-2+ T cells in the mouse have been investigated. In short-term in vivo homing studies, Lyt-2- T cells localized consistently more efficiently than Lyt-2+ T cells in Peyer's patches (about 1.5 times as well), whereas both populations localized roughly equivalently in peripheral lymph nodes. These homing characteristics of Lyt-2- and Lyt-2+ subsets are largely independent of their organ source. The specificity of migration appears to be determined by selective recognition of organ-specific determinants on the endothelial cells of high endothelial venules (HEV), specialized venules that mediate the exit of migrating lymphocytes from the blood: In an in vitro assay of lymphocyte binding to HEV in lymphoid organ frozen sections, Lyt-2- cells constituted a significantly and consistently greater proportion of T cells binding to Peyer's patch HEV than of those binding to peripheral node HEV. The homing and HEV recognition preferences of the Lyt subsets are reflected in differences in their in situ representation in mucosal vs nonmucosal lymphoid organs, which suggests that the selective migration of these populations may be an important factor in determining the character of local immune responses.

Animals↗
PubMed1987

Enhancement of therapeutic effects of recombinant interleukin 2 on a transplantable rat fibrosarcoma by the use of a sustained release vehicle, pluronic gel.

We have tested the feasibility of pluronic F-127 gel (PLF-127; a polyoxyethylene-polyoxypropylene surface active block copolymer) as a sustained release vehicle for topical administration of interleukin 2 (IL-2) in order to enhance the therapeutic effects of IL-2 against a rat fibrosarcoma, KMT-17. Injection of human DNA recombinant IL-2 (3 X 10(4) units s.c.) in 30% (w/w) PLF-127 into rats provided detectable serum IL-2 levels for up to 10 h, while injection of IL-2 alone provided detectable IL-2 levels for 3 h. When, following s.c. inoculation with 1 X 10(5) KMT-17 tumor cells into rats, IL-2 (6 X 10(4) units/day) in PLF-127 gels was injected s.c. around the growing tumor inoculum every 2 days for 10 days from Day 1 to Day 19, the survival days of rats were more prolonged [mean survival day, 32.3 +/- 5.4 (SD)] as compared with that of rats treated with saline [20.7 +/- 2.1] than mean survival days of rats treated with IL-2 alone [27.3 +/- 4.5] or PLF-127 alone [22.9 +/- 3.3]. Moreover, the span of mean survival days of rats treated with IL-2 in PLF-127 locally (31.7 +/- 5.9) was much longer than that of rats given IL-2 in PLF-127 systemically (22.8 +/- 3.4). By means of a Winn assay, stronger tumor neutralizing activities were observed in regional lymph node cells obtained from tumor bearing rats treated with IL-2 in PLF-127 than were observed in lymph node cells from rats treated with IL-2 alone or PLF-127 alone (percentage of inhibition, 90.3, 12.2, and -15.5%, respectively). The therapeutic effects of IL-2 were thus found to be consistent with the antitumor activity in regional lymph node cells. These results suggest that the enhanced therapeutic effects of IL-2 in PLF-127 are due to enhancement of antitumor immune responses induced by sustained IL-2 activity at the tumor sites.

Animals↗