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Results for “Local Lymph Node Assay”

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PubMed2001

Genetic detection of lymph node micrometastases in patients with gastric carcinoma by multiple-marker reverse transcriptase-polymerase chain reaction assay.

BACKGROUND: Some patients with gastric carcinoma experience local disease recurrence despite undergoing curative resection of the tumor and regional lymph nodes (LNs), suggesting the presence of occult micrometastases. To evaluate the presence of gastric carcinoma micrometastasis in patients with otherwise histologically negative LNs, the authors established and tested a multiple-marker reverse transcriptase-polymerase chain reaction (RT-PCR) assay. METHODS: The authors assessed 435 LNs from 28 patients with gastric carcinoma who underwent gastrectomy with lymphadenectomy using the multiple-marker RT-PCR assay in addition to histologic examination. Carcinoembryonic antigen (CEA), cytokeratin-20 (CK-20), and MAGE-3 gene markers were used in this assay. LNs were scored positive for metastasis if at least one marker was positive. The presence of LN micrometastases also was verified by immunohistochemistry in histologically negative and RT-PCR positive LNs. RESULTS: Sixty-nine of 435 LNs (16%) were positive for CEA (12%), CK-20 (10%), or MAGE-3 (5%). None of 16 control LNs obtained from disease free patients was positive by RT-PCR assay. Of 414 histologically negative LNs, 50 LNs (12%) were scored as positive for metastasis by the assay. Of 26 patients who underwent curative resection, the disease stage was upgraded in 10 patients by genetic diagnosis (from Stage IA to Stage IB in 5 patients, from Stage IB to Stage IIIA in 2 patients, from Stage IB to Stage IV in 1 patient, from Stage IB to Stage II in 1 patient, and from Stage II to Stage IIIB in 1 patient). In the latter 10 patients, immunohistochemistry identified LN micrometastases in 4 patients. Two patients with micrometastasis by genetic diagnosis had recurrent disease within 1 year. CONCLUSIONS: The current results indicate that the multiple-marker RT-PCR assay is a useful tool for the detection of micrometastases in regional LNs in patients with gastric carcinoma and may improve the staging system of gastric carcinoma for these patients.

Adult↗
PubMedDate not supplied

Molecular detection of micrometastases and circulating tumor cells in melanoma prostatic and breast carcinomas.

The molecular detection of circulating tumor cells (CTC) and micrometastases may help develop new prognostic markers in patients with solid tumors. In the last 10 years, numerous groups have attempted the detection of occult tumor cells in solid malignancies using the highly sensitive reverse transcriptase polymerase chain reaction (RT PCR) technique. These assays were in the vast majority directed against tissue specific markers. In most studies on prostatic carcinoma, RT PCR was able to specifically detect prostatic tissue specific markers in the peripheral blood (PB), bone marrow (BM) and lymph nodes of patients with localized and metastatic disease. Melanoma related transcripts were detected by RT PCR in the PB, BM and lymph nodes of patients with localized and advanced tumors. In most studies, melanoma related markers were shown to be specific except when assayed in lymph nodes. RT PCR positivity rates were highly variable between studies. Despite tFlese discrepancies, many authors have shown a statistically significant correlation between RT PCR positivity and a poorer outcome in both melanoma and prostatic carcinoma. In breast carcinoma, all markers that have been extensively tested were shown to be non-specific. Because of the many limitations of RT PCR (e.g. false positives), many groups are developing new approaches for the detection occult tumor cells. One of these techniques involves immunobead isolation of CTC and micrometastases prior to down stream analysis. The tumor rich magnetic fraction can be subjected to RT PCR, immunocytochemistry and flow cytometry. In conclusion, the molecular detection of occult tumor cells in solid tumors seems very promising and the techniques used for this purpose are in continuous evolution. Large prospective and interlaboratory variability studies are necessary to determine the accuracy and prognostic value of these assays.

Breast Neoplasms↗
PubMed1989

Active specific chemoimmunotherapy of lymph-node metastasis from a poorly immunogenic murine fibrosarcoma.

The fibrosarcoma MCA-SP, which was recently induced with methylcholanthrene (MCA) in C3H/HeJ mice, displays poor immunogenicity in in vivo prophylaxis. A cell variant MCA-SPN1, which bears a tumor-specific transplantation antigen (TSTA) cross-reactive with the parental line MCA-SP, was selected because of its proclivity for axillary lymph-node metastases. Although these lymph-node metastases were resistant to sinecomitant (post-excisional) immunity, they were susceptible to combined active and passive specific chemoimmunotherapy, using tumor-specific, 1-butanol-extracted, preparative isoelectric focusing-purified, TSTA (1 microgram weekly sc injections), cyclophosphamide (CY, a single intraperitoneal 20 mg/kg dose), and adoptive transfer of immune splenic T lymphocytes, which had been re-stimulated in vitro with extracted TSTA and interleukin-2. This triple regimen both reduced the incidence of spontaneous lymph-node metastases, and prolonged the survival of tumor-bearing, as well as tumor-resected hosts. The results from local adoptive transfer assay using T-lymphocyte subpopulations of spleen and lymph nodes in these treated hosts suggested that Lyt 2+ cytotoxic T-lymphocytes (CTL) mediated in vivo tumor-neutralization. Thus TSTA/CY/CTL therapy activates tumoricidal host responses effective against the poorly immunogenic MCA-SP tumor and its lymph-node metastases.

Animals↗
PubMed1997

Preliminary characterization of an immunosuppressive inducer factor secreted by the JEG-3 choriocarcinoma cell line: in vitro and in vivo studies.

PROBLEM: The direct immunosuppressive and suppressive inducing capacities of supernatants from human trophoblastic choriocarcinoma cell lines (HCS) are well investigated in several former studies. The responsible factor is not yet determined. METHOD OF STUDY: We first confirmed those data and we purified a 3-5-kDa suppressor-inducer factor from HCS by using high performance liquid chromatography (HPLC) on both DEAE and gel filtration columns, followed by ultrafiltration. We then tested the activities of such isolated fractions on in vitro immune responses from human cells and in vivo by its effects in a murine local graft-versus-host (GVH) assay (popliteal lymph node assay, PLN). RESULTS: A single fraction induces both "direct suppression" in vitro as well as in vitro suppressor cell activation/development in human peripheral blood lymphocyte cultures as assessed by suppression of cells cultured in such a fraction containing culture medium of the mixed lymphocyte reaction. Furthermore the very same fraction suppresses in vivo murine allogeneic immune responses as assessed by a local GVH reaction (PLN assay). CONCLUSIONS: We have isolated a suppressive fraction, whose activities suggest that it might be of interest not only in reproductive immunology, but also in transplantation systems.

Animals↗
PubMed1992

Frequency of antigen-specific B cells during experimental ocular Chlamydia trachomatis infection.

Chlamydia-specific antibody-secreting cells have been identified in conjunctiva and draining cervical lymph nodes by an ELISPOT assay in a cynomolgus monkey model of trachoma. These local sites contained numbers of chlamydia-specific B cells that were higher than those in distant inguinal lymph nodes and peripheral blood. The numbers of chlamydia-specific immunoglobulin G-secreting B cells observed were 5 to 57 per 10(6) cells in conjunctiva and 24 to 996 per 10(6) cells in cervical lymph nodes during conjunctival infection or after challenge of immune monkeys with the chlamydial 57-kDa heat shock protein (hsp60). These studies demonstrate a large chlamydia-specific B-cell component in the conjunctiva during ocular chlamydial infection. These results are similar to our findings for chlamydia-specific T-cell responses.

Animals↗
PubMed1988

The effects of bacterial lipopolysaccharide (LPS) on high endothelial venules and interdigitating cells in mouse lymph nodes.

Migration of lymphocytes into lymph nodes is directly controlled by interaction of lymphocytes with the high endothelial venules (HEV), located in T cell-dependent areas of lymph nodes. Little is known about the mechanisms that regulate the activity of the HEV and the specificity of interaction with lymphocytes in various lymphoid organs. It has been suggested that the non-lymphoid interdigitating cells (IDC) play a role in maintaining the integrity of T cell areas. We therefore wished to determine whether the function of the HEV could be influenced by disturbing the integrity of the T cell area. This was assayed in mice by injecting bacterial lipopolysaccharide (LPS) in the hind footpad. The T cell-dependent areas of mouse popliteal lymph node were studied using immunohistochemistry, and attention was paid to the HEV specificity using short-term in vivo localization assays and in vitro lymphocyte binding assays. After LPS administration, apparent changes in the number of interdigitating cells and T cells in the draining lymph node were found. In addition, interdigitating cells lost their typical dendritic appearance. However, after LPS treatment, neither size, localization nor receptor specificity for lymphocyte subsets of HEV differed from control HEV. Although administered subcutaneously in the footpad, the effect of LPS on T cells and IDC was much more dramatic in the spleen than in the draining lymph nodes. This points to a difference in the processing of the endotoxin in the two organs.

Animals↗
PubMed1981

Local cellular immunity in the draining lymph nodes of mice after immunization with histocompatibility alloantigens.

Mice immunized with allogeneic cells exhibit a local component of cellular immunity that is confined to lymph nodes draining the site of immunization. After immunization of C3H mice by skin grafting or by i.p. or footpad injection of F1 hybrid spleen cells, draining lymph nodes show increased responsiveness toward the immunizing histocompatibility determinants for at least 3 months after immunization. This local component of immunity is not detected in a traditional graft-versus-host (GVH) assay of splenomegaly. To demonstrate this immunity, we used a GVH assay in which pieces of whole lymph nodes were grafted onto F1 hybrid host kidneys. The enlargement of each grafted node piece is taken as the measure of its immunological responsiveness. We interpret our results to indicate that this node-onto-kidney assay detects the contribution of a relatively immobile donor cell population that exerts its effects in lymph nodes local to, but not distant from, the site of immunization.

Animals↗
PubMed1992

Localization of inhaled trimellitic anhydride to lung with a respiratory lymph node antibody secreting cell response.

Male Sprague-Dawley rats were exposed to trimellitic anhydride (TMA) by inhalation (500 micrograms/m3), 4 hours a day, for 1 to 10 days. TMA was localized to lung cells by immunoelectron microscopy. Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis were used in an attempt to localize TMA to lung lavage proteins. The lung-associated lymph node (LALN) B-lymphocyte response was measured by quantitation of immunoglobulin (Ig)G, IgA, and IgM antibody secreting cells specific for TMA rat serum albumin (TM-RSA) by use of the enzyme-linked immunospot assay (ELISPOT) method. The IgG, IgM, and IgA antibody response to TM-RSA in serum and lavage fluid was quantitated by ELISA. Lung injury was assessed by the number of external lung hemorrhagic foci and lung weight. Immunoelectron microscopy localized TMA to alveolar and bronchial cells on all exposure days. ELISA detected trace amounts of TMA haptenized lavage proteins that could not be detected by Western blot analysis. A marked increase occurred in lung injury from day 7 to 10. The LALN IgG, IgA, and IgM antibody secreting cell response to TM-RSA paralleled measures of lung injury. IgG, IgM, and IgA serum and lavage antibody to TM-RSA were correlated with lung injury measures. Lavage and serum IgG antibody levels had the highest correlation with lung injury.

Administration, Inhalation↗
PubMed1993

Lethal systemic graft-vs-host disease in neonatally tolerant, but not in F1 hybrid mice.

The possibility, conditions, and quantitative aspects of eliciting GVHD in CBA (H-2k) mice made neonatally tolerant to the alloantigens of the donor A (H-2a) strain were studied. The intravenous injection of different doses (10(7)-2 x 10(8)) of A spleen cells caused a severe, often fatal, systemic GVHD in 12-month-old tolerant mice. The GVHD was found to be specific: spleen cells of a third party strain (B10) did not induce any disease. The intensity and the mortality of the GVHD depended on the cell dose and on the age of the recipients. In contrast, unirradiated (CBA x A)F1 recipients proved to be resistant to the lethal disease. In spite of their different susceptibility to the systemic GVHD, the tolerant and F1 hybrid recipients showed equally strong local GVH reactivity in the popliteal lymph node enlargement assay. Neonatally tolerant mice offer a new, sensitive model for the induction of lethal GVHD without the need of immunosuppression or irradiation.

Aging↗
PubMed1984

Results of the local graft-versus-host reaction support the immunogenetic concept of the insulin-dependent Diabetes mellitus in BB rats.

The local GVHR was studied by the popliteal lymph node assay in adult randombred BB/DK rats, their parental WOK rats and their interstrain F1 and BC1 hybrids as well as in the LEW triplet of congenic and recombinant strains. A strong GVHR was observed when BB/DK rats were used as recipients. On the contrary, only weak GVHR were found in most cases with BB/DK donors of lymphoid cells. This lowered graft-versus-host reactivity of BB lymphoid cells provided further evidence for a genetically determined defect of the differentiation process of lymphoid cell subpopulations, most probably caused by the mutant RT1u haplotype in BB rats.

Animals↗
PubMed1981

Effect of local anaesthetics on immune reactivity.

Two local anaesthetics, pentacaine and trimecaine, were assayed for their effects on immune reactivity of mice in the regional popliteal lymph node after local injection of the antigen, on the regional graft-versus-host reaction and on the ability to reject skin allografts. These local anaesthetics exhibited a dose-dependent, immunosuppressive effect on all the systems used, although the mechanism of their action was different: pentacaine exerted a destructive effect on lymphocytes, whereas trimecaine induced a blockade of the lymphocyte functions.

Acetanilides↗
PubMed1978

Local graft-versus-host reaction in mice evoked by Peyer's patch and other lymphoid tissue cells tested in a lymphocyte-induced angiogenesis assay.

The competence of murine Peyer's patch cells to evoke a local GVH reaction was compared with that displayed by lymphoid cells of spleen, lymph nodes, thymus, bone marrow, peritoneal cavity and peripheral blood. The local GVH reaction was assessed in a lymphocyte-induced angiogenesis assay in which an intradermal injection of lymphoid cells results in a new blood vessel formation at the injection site, and the number of vessels corresponds to the number of the immunocompetent cells injected. Peyer's patch cells were capable of mounting a local GVH reaction of intensity comparable to that evoked by the corresponding number of thymus cells or a four times lower number of spleen cells. The highest activity was exhibited by lymph node and peripheral blood cells while bone marrow cells were the least active. A striking increase in angiogenic response was observed after X-irradiation of the recipients with 700R.

Animals↗
PubMed1996

European Lyme borreliosis: 231 culture-confirmed cases involving patients with erythema migrans.

In 1994, we isolated Borrelia burgdorferi sensu lato from 231 patients with erythema migrans who presented to the University Medical Center in Ljubljana, Slovenia. Samples of erythema migrans-affected skin were placed in media to support the growth of Borrelia species and evaluated in Ljubljana and Chicago. Patients whose cultures were positive included 132 women and 99 men; 136 of these 231 patients recalled a tick bite. Patients noted a rash an average of 24 days after a bite and presented a mean of 34 days after the bite with erythema migrans (mean diameter. 16 cm). Itching (44%) burning (18%), and pain (11%) were the most common local symptoms. Systemic complaints (40%) included headache, fatigue, malaise, and arthralgia. Other than erythema migrans, findings on physical examination were minimal (< 5% had fever, and in < 10% local lymph nodes were affected). Serial serological studies using indirect immunofluorescence assay, ELISA, and Western blot methods were performed, and antibodies to B, burgdorferi sensu lato were detected in < 50% of samples from patients. This is the largest series reported to date of patients with culture-confirmed Lyme borreliosis. It highlights the deficiencies of serological tests in early disease, demonstrates the sensitivity of direct detection methods for evaluation of patients with erythema migrans, and suggests that patients with early Lyme borreliosis in Slovenia may suffer a milder illness than those in the United States.

Adolescent↗
PubMed1979

Subpopulations of Ig-secreting cells induced by peroxidase immunization: discrimination according to antibody storage and secretion.

Mice were injected in their hind footpads with peroxidase (PO) emulsified in Freund's complete adjuvant. The development of cells secreting anti-peroxidase antibody (Ab) and cells secreting immunoglobulins (Ig) were detected in the draining popliteal lymph nodes in the subsequent 35 days, using local haemolysis plaque assay with sheep red cell blood cells coated with either PO or anti-mouse Ig antibody. Plaque-forming cells (PFC) were isolated from the centre of plaques by micromanipulation and after appropriate treatment, were examined by electron microscopy for their intracellular Ab content and in corporation of [3H]-thymidine. Four subpopulations of Ig secreting cells were distinguished: (1) cells secreting Ig without Ab function and not containing intracellular Ab detectable between days 5 and 20; (2) cells secreting Ig without Ab function but containing Ab appearing on day 6 and present throughout the immune response; (3) cells secreting Ab and containing Ab; (4) cells secreting Ab, but without detectable intracellular Ab. These last subpopulations appeared on day 7 and were found in all subsequent assays. The analysis of the kinetics of these subpopulations suggest that cells secreting Ig without Ab function might be precursors of Ab secreting cells.

Animals↗
PubMed1979

Functional differentiation of mouse T lymphocytes. GVHR-precursors: tissue origin and specificity of activity inducer.

For theoretical and practical reasons, it is important to find out whether the differentiation of T cell precursors to the functional lymphocytes can be induced under in vitro conditions. Using the local GVHR assay (based on the enlargement of the popliteal lymph node), the inducibility of the precursors of reactive cells was studied with bone marrow, thymus, spleen, and lymph node cell suspensions submitted to short-term incubation with cell-free extracts from calf thymus, spleen or brain. GVHR-precursors from bone marrow were inducible not only specifically (i.e., with thymus extract) but also--and even to a higher degree--with spleen or brain extract. Thymus and spleen cell suspensions (the latter also depleted of the reactive subpopulation by treatment with anti-Thy 1.2 serum and complement) were, on the other hand, inducible mainly specifically, whereas lymph node cells were refractory to induction. The inductive action of tissue extracts obviously depends on the tissue origin of T cell precursors; their effects on pre- and postthymic differentiation of T lymphocytes are discussed.

Animals↗
PubMed1976

Appearance of antibody-forming cells in lymphocytes from the lower respiratory tract of the dog after intrapulmonary or intravenous immunization with sheep erythrocytes.

To investigate the mechanisms of expression of humoral immunity in the lower respiratory tract, dogs were immunized either locally (intrapulmonary) or systemically (intravenous) with low doses of sheep erythrocytes. Bronchoalveolar cells obtained by pulmonary lavage and hilar lymph nodes were assayed during the primary immune response for their concentrations of antibody-forming cells with the Jerne plaque technique. Antibody-forming cells consistently appeared among bronchoalveolar cells after intravenous immunization but failed to accumulate in this cell population after local administration of the identical dose of sheep erythrocytes. By contrast, after local immunization, antibody-forming cells appeared in the hilar lymph nodes in generally greater concentrations than after intravenous immunization. The results demonstrated that the anatomic site of presentation of this particulate antigen to the lung (intravascular versus intra-alveolar) as well as the dose of antigen are important determinants of the distribution of antibody-forming cells in these 2 pulmonary lymphoid tissues. The data suggested that bronchoalveolar lymphocytes and hilar lymph node cells function independently in the expression of humoral immunity in the lower respiratory tract.

Animals↗
PubMed2000

The fungal cell wall component beta-1,3-glucan has an adjuvant effect on the allergic response to ovalbumin in mice.

The polyglucose beta-1,3-D-glucan is a major structural component of the cell wall of yeasts and fungi. In the present study, the adjuvant activity of beta-1,3-glucan from the fungus Sclerotinia sclerotiorum (SSG) on the response to the model allergen ovalbumin (OA) was studied, using the popliteal lymph node assay (PLNA) in BALB/c mice. The adjuvant activity on the local cellular response was determined by measuring the weight, cell number, and proliferation of the extracted PLNs. The levels of OA-specific immunoglobulin (Ig)E, IgG1, and IgG2a in serum were measured by enzyme-linked immunosorbent assay (ELISA). Groups of 8 mice were given either SSG + OA, SSG alone, or OA alone on d 0. Thereafter they were exsanguinated on d 20, or reinjected with OA on d 21, before exsanguination on d 26 or 33. Only on d 26 was SSG + OA found to significantly increase the PLN weight and cell numbers, but not cell proliferation (thymidine incorporation), compared with OA or SSG alone. SSG + OA was also found to significantly increase both the anti-OA IgE and IgG1 levels on d 20, 26, and 33 compared to OA alone. Compared to SSG alone, SSG + OA increased the OA-specific IgE and IgG 1 levels significantly on d 26 and 33, but not on d 20. A similar increase was not found for IgG2a. Our results show that beta-1,3-D-glucan provides a clear Th2-dependent (allergic) immune response to OA, indicated by elevated levels of IgE and IgG1 and not IgG2a, in the mouse model used.

Adjuvants, Immunologic↗
PubMed1998

Measurement of serum monocyte chemoattractant protein-1 and its clinical application for estimating the activity of granuloma formation in sarcoidosis.

BACKGROUND AND AIM OF THE WORK: The role of monocyte chemoattractant protein-1 (MCP-1) in bronchoalveolar lavage fluids from sarcoidosis patients was previously reported. To study the role of MCP-1, we evaluated the serum MCP-1 and its clinical significance in sarcoidosis. METHODS: The serum MCP-1 level was measured in 47 patients with sarcoidosis and 10 normal healthy controls with the use of an enzyme-linked immunosorbent assay. The localization and mRNA expression of MCP-1 in sarcoid lymph nodes were evaluated by an immunohistochemical method using an anti-MCP-1 monoclonal antibody and an in situ hybridization technique to determine the cellular source(s) of MCP-1. RESULTS: Serum MCP-1 levels were significantly elevated in the sarcoidosis patients compared with the healthy controls (698.3 +/- 101.9 vs. less than 39 pg/ml, p < 0.001). A comparison of the patients' serum MCP-1 levels among standard radiographic stages revealed that the serum MCP-1 was significantly higher in early stages: stage 0 vs. III, and stage I vs. II. In addition, the serum MCP-1 levels were significantly correlated with the serum angiotensin converting enzyme levels (r = 0.539, p = 0.0006). MCP-1 expression was detected in macrophages peripheral to the epithelioid granuloma in sarcoid lymph nodes, by both immunohistochemistry and in situ hybridization. CONCLUSIONS: These data suggest that MCP-1 may be expressed by the macrophages in the granuloma throughout the body, and that the measurement of serum MCP-1 levels may have clinical value as an indicator in estimating the activity of granuloma formation throughout the body in sarcoidosis.

Adult↗