Local lymph node assay: differentiating allergic and irritant responses using flow cytometry.

The murine local lymph node assay (LLNA) is a method for assessing the contact sensitization potential of chemicals. Based on events that occur during the induction phase of a contact sensitization response, the LLNA measures the in vivo proliferation of cells in the draining lymph nodes (DLNs) of mice following topical exposure to chemicals. In terms of predictive identification of important skin sensitizers, the LLNA has been shown to be at least as sensitive as, and much more reliable than, current guinea pig tests. However, proliferation has also been observed following treatment with some irritants. In an attempt to distinguish allergic from irritant-induced proliferation, flow cytometric techniques have been used to examine the phenotype of lymphocyte subsets in the DLNs as well as markers of T-lymphocyte activation and memory. Mice were treated on the ears for 3 consecutive days with allergens or irritants. The DLNs were harvested 72 h after the final treatment. Single-cell suspensions were prepared, counted, and stained for analysis of the percentages of T cells and B cells and T-cell expression of two adhesion molecules that have been associated with differentiating na?ve and activated/memory T cells, CD62L (L-selectin) and CD44 (H-cam). Increases in lymph node cellularity were observed in both allergen- and irritant-treated mice relative to na?ve and vehicle-treated animals. Mice treated with allergens showed a preferential increase in the percentage of B220(+) B cells compared with irritant-treated mice. Treatment with allergens, but not irritants, resulted in a selective increase in the percentages of CD4(+) and CD8(+) cells expressing the T-cell activation/memory phenotype CD62L(lo)CD44(hi). Taken together, flow cytometric analysis of cell phenotype and expression of T-cell activation/memory markers may provide important information for differentiating allergen- and irritant-induced proliferative responses in the DLNs of chemically treated mice.     

Popliteal lymph node (PLN) assay to study adjuvant effects on respiratory allergy

Different variants of the popliteal lymph node (PLN) assay have been published. Here we describe the adjuvant popliteal lymph node assay, an immune response assay to study the adjuvant activity of soluble substances as well as particulate matter. The substance to be studied for adjuvant activity is injected into the hind footpad of mice or rats together with an antigen. Adjuvant activity is determined as the increase in PLN weight and cell numbers in animals receiving antigen together with the substance under study, compared with PLN weight and cell numbers in animals given the antigen without the substance in question, and animals given the putative adjuvant alone. Because lymph node weight and cell numbers are immunologically non-specific parameters, specific immune response assays like serum antibody responses or antibody-forming cell numbers should additionally be performed. Different antigens and immune response assays may be used, depending on the research question asked. In relation to respiratory (or food) allergy, the assays should as a minimum include determination of specific IgE in serum, and preferably also IgG1 (mouse). Serum specific IgG2a antibody determination may be added to get an indication of the Th1-Th2-balance of the response. The adjuvant PLN assay, with cellular response assays performed in the draining popliteal lymph node and antibody determinations in serum, requires small amounts of test material. The assay offers a practical, sensitive and reproducible method to determine the adjuvant activity of soluble substances as well as particulate material, with the possibility to also perform mechanistic studies.     

Ontogeny of the antigen-reactive lymph follicle-forming capacity of the popliteal lymph node in neonatal mice

The ontogenetic development of the reactive lymph follicle-forming capacity of the popliteal lymph node was investigated immunohistochemically in young mice which had received a single injection of hemocyanin (KLH) in a rear footpad at a predetermined age (between 1 and 21 days). The mice were sacrificed at various intervals after injection. In non-stimulated young mice, primary lymph follicles first appeared in the popliteal node at 11 days of age. When KLH was given to 7-day-old or older mice, each draining popliteal node showed a marked increase in B lymphocytes in the extrafollicular zone 3 days after injection and produced a number of "new" lymph follicles outside the pre-existing follicles over the next few days. In mice injected at 2-4 days of age, these nodes showed an increase in B lymphocytes in the outer cortex and had produced several lymph follicles by 8 days of age. The number of lymph follicles produced by each node tended to increase in line with age at injection. These results indicate that neonatal popliteal nodes become able to produce lymph follicles in response to exogenous antigens some time before ontogenetically developing follicles appear. The formation of new lymph follicles observed in draining popliteal nodes after KLH injection at an early postnatal age is discussed in relation to the ontogenetic development of stromal cells (precursors of follicular dendritic cells) that are capable of interacting with B lymphocytes and the extent of B lymphocyte influx into the node induced by KLH stimulation.     

Early detection of Leishmania (Leishmania) chagasi in draining lymph node after subcutaneous inoculation in hamster

To study the route of migration of Leishmania (Leishmania) chagasi to the viscera from the subcutaneous inoculation site, ultrastructural studies were carried out. These studies on popliteal draining lymph nodes of hamsters inoculated with 10⁷ promastigotes of Leishmania (Leishmania) chagasi in the hind footpad were carried out. Parasites were detected in lymph nodes just 2 h after inoculation. Parasites were either preserved or degenerated in macrophages. In 24 h signs of binary division of the parasites were found in macrophages. This fact suggests that the parasites migrate from skin to the lymph node early in the infection within phagocytes and proliferate in lymph nodes before dissemination to the viscera.     

Cytoplasmic immunoglobulins in giant lymph node hyperplasia (Castleman's tumour)

The immunohistologic features were studied in 6 cases of giant lymph node hyperplasia (GLNH) and the cytoplasmic immunoglobulin (CIg) characteristics were compared with those of follicular lymphoma and non-specific follicular lymph node hyperplasia. By use of the peroxidase - antiperoxidase (PAP) technique it was shown that GLNH comprised a mosaic, polyclonal population of CIG-producing cells, the CIg pattern being comparable with that observed in follicular lymphadenitis. In contrast, follicular lymphomas disclosed a definite monoclonal pattern, the cytoplasmic Ig containing only one light chain of kappa type. This led to the conclusion that GLNH is not a neoplastic change, but has the characteristics of a reactive process within the B-cell compartment.     

Comparison of liposome immune lysis assay (LILA) and enzyme-linked immunosorbent assay (ELISA) for detection of antibodies

Anti-α-chymotrypsinogen A antibody was assayed by both enzyme-linked immunosorbent assay (ELISA) and liposome immune lysis assay (LILA). The detection limit was slightly affected by the measurement conditions in ELISA; however, it was possible to control the detection limit and to achieve a lower level by adapting the measurement conditions in LILA. LILA is believed to offer a simple and highly sensitive method for measuring the concentration of antibody in serum.     

Interaction of rabbit lymph node cells with bacteria (Salmonella paratyphi B)

In five rabbits immunized withSalmonella paratyphi B antigen in all four paws, incubation of the regional lymph node cells with the same bacteria was followed by a significant shift of some of the bacteria from the supernatant of the culture medium to the sediment (as compared with the controls). In six rabbits, bactericidal activity was demonstrated in extracts prepared from the regional lymph nodes only 30 hours after immunization. No antibodies were present in extracts from control rabbits up to the fifth day after immunization.     

Chemiluminometric biochemical induction assay (CBIA) for the detection of DNA-damaging agents

A microbroth chemiluminometric version of the biochemical induction assay (BIA) was developed using a chemiluminescent substrate widely used to detect beta-galactosidase in high-throughput screening (HTS) laboratories. The assay was run in both 96-well and 384-well plate formats using the Zymark RapidPlate liquid handling system to transfer samples and reagents. Chemiluminescence was read using the Victor-2 multilabel counter. The new microbroth chemiluminometric method, the CBIA, allowed rapid screening of samples, crude extracts, and pure compounds for their DNA-damaging effects in bacteria. In screening a small subset of our natural products library samples by the agar plate BIA and the CBIA, the latter yielded a higher hit rate, suggesting it is more sensitive than the agar plate assay. The CBIA was unaffected by the colored samples often encountered during screening of crude natural products extracts.     

Minimum acceptable sensitivity of intraoperative gamma probes used for sentinel lymph node detection in melanoma patients

The aim of this study was to determine the suspension level for the sensitivity of an intraoperative scintillation gamma probe in the detection of the sentinel lymph node (SLN) in melanoma patients.Thirty-eight consecutive patients with melanoma were enrolled in the study during a 12-month period and underwent lymphatic scintigraphy after the peritumoral intradermal administration of about 14 MBq of ^99mTc-nanocolloids. The SLNs were successfully removed during the surgical intervention about 4 h later.To identify and localize the SLN, a scintillation NaI(Tl) collimated probe was used. Predictably, the probe sensitivity decreased as the photopeak energy window was progressively narrowed, from 6.9 ± 0.7 counts per second (cps)/kBq (designated as the ‘optimum,’ or ‘OPT,’ sensitivity) to 2.5 ± 0.3 cps/kBq (LOW sensitivity) and to 1.4 ± 0.2 cps/kBq (VLOW sensitivity).Maximum lymph node count rates (cps) were determined for the foregoing energy windows prior to skin incision (PRE_OPT, PRE_LOW, PRE_VLOW, respectively) and in vivo after incision (INV_OPT, INV_LOW, INV_VLOW).Forty-three SLNs were removed with a mean source-to-detector distance of 46 ± 24 mm (min 12 mm, max 92 mm). Four SLNs could not have been detected using PRE_LOW. This figure would have decreased to 34, with nine undetectable lymph nodes, with PRE_VLOW. One SLN could not have been identified using INV_LOW and four could not have be identified using INV_VLOW.In the clinical scenario of SLN detection in melanoma patients, a system sensitivity of 2.5 cps/kBq represents a suspension level, that is, a level under which the equipment must be suspended from clinical use and the poor performance must be investigated.