Local lymph node assay (LLNA) for detection of sensitization capacity of chemicals

The local lymph node assay (LLNA) is a murine model developed to evaluate the skin sensitization potential of chemicals. The LLNA is an alternative approach to traditional guinea pig methods and in comparison provides important animal welfare benefits. The assay relies on measurement of events induced during the induction phase of skin sensitization, specifically lymphocyte proliferation in the draining lymph nodes which is a hallmark of a skin sensitization response. Since its introduction the LLNA has been the subject of extensive evaluation on a national and international scale, and has been successfully validated and incorporated worldwide into regulatory guidelines. Experience gained in recent years has demonstrated that adherence to published procedures and guidelines for the LLNA (e.g., with respect to dose and vehicle selection) is critical for the successful conduct and eventual interpretation of the data. In addition to providing a robust method for skin sensitization hazard identification, the LLNA has proven very useful in assessing the skin sensitizing potency of test chemicals, and this has provided invaluable information to risk assessors. The primary method to make comparisons of the relative potency of chemical sensitizers is to use linear interpolation to estimate the concentration of chemical required to induce a stimulation index of three relative to concurrent vehicle-treated controls (EC3). In certain situations where there are available less than optimal dose response data a log-linear extrapolation method can be used to estimate an EC3 value which can reduce significantly the need for repeat testing of chemicals. The LLNA, when conducted according to published guidelines, provides a robust method for skin sensitization testing that not only provides reliable hazard identification information but also data necessary for effective risk assessment and risk management.     

Ultrastructural organization of the endotheliocytes of iliac lymph node capillaries in pregnancy (morphometric study)

Using light and electron microscopy and morphometry, the morphological changes in the lymph nodes of arterial and venous parts of capillaries were studied on the 11th, 17th and 21st days of pregnancy in rats. Ultrastructural changes in endothelial cells of blood vessels in the uterine lymph nodes during normal pregnancy are of adaptive nature and are possibly responsible for the relief of the blood congestion in the system of the inferior vena cava and for the improvement of the utero-placental circulation.     

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.     

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.     

Sensitivity of lymph node aspiration in localized cutaneous leishmaniasis due to Leishmania (Viannia) braziliensis.

Twenty nine patients with localized cutaneous leishmaniasis had lymph node and skin ulcer aspirations for culture of Leishmania with the modified Marzochís vacuum aspiratory technique. Sensitivity of lymph node aspiration was 58.6% and 34.5% for skin ulcer aspiration (P=0.06). Combined sensitivity of the two methods was 79.3%. There was no agreement between methods (Kappa Index = -0.084; CI95% -0,45; 0,28) showing the potential complementary roles in diagnostic approach.     

Cyclophosphamide inhibits antibody-dependent cellular cytotoxicity (ADCC) suppression exerted by lymph node cells

The effect of cyclophosphamide (Cy) on suppression of antibody-dependent cellular cytotoxicity (ADCC) by lymph node cells (LNC) was evaluated. The results show that the suppression of ADCC exerted by LNC was abrogated when mice had been treated with Cy. Moreover, it was shown that ADCC inhibition induced by LNC was mediated by soluble factor(s) and that treatment with a single dose of 200 mg/kg ip of Cy, significantly decreased its release. In addition, suppressor activity of normal LNC was enriched by depletion of adherent cells and was not affected by treatment with monoclonal anti-Thy 1.2 plus complement. These observations indicate that modulatory cells are nonadherent and lack characteristic T-cell markers. Thus, we conclude that this suppressor system, which normally controls ADCC activity, can be inhibited by treatment of mice with Cy and that this effect may explain the enhancement of ADCC observed in splenocytes of Cy-treated animals.     

Studies on the role of lymphocyte-activating factor (Interleukin 1) in antigen-induced lymph node lymphocyte proliferation

The in vitro proliferation of primed lymph node lymphocytes (LNL) in response to the soluble antigen ovalbumin (OVA) was dependent upon the presence of adherent cells. Restoration of OVA-induced LNL proliferation could be achieved by addition of highly purified lymphocyte-activating factor (LAF; Interleukin 1, IL 1): LAF (IL 1) did not stimulate LNL proliferation in the absence of the priming antigen or T lymphocytes. Furthermore, treatment of the LNL with antimacrophage serum completely blocked the ability of the LNL to respond to OVA and LAF (IL 1), suggesting that the residual macrophages in the LNL population were necessary to provide an additional function or signal, possibly antigen presentation, in conjunction with LAF (IL 1). These data therefore support the two signal hypothesis of macrophage-mediated lymphocyte activation and demonstrate the ability of LAF (IL 1) to provide one of these signals.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. III. Cholera enterotoxin induces lymph node cells to release soluble factor(s) which enhance(s) antibody synthesis by antigen-treated lymph node cells.

Unprimed or KLH-primed rabbit lymph node cells were pulsed with cholera enterotoxin or KLH for 2 hr and washed. KLH-treated LNC were mixed with equal numbers of CT-treated LNC or boiled CT-treated LNC. Cocultivation of CT-treated LNC with KLH-treated cells resulted in at least a 100% increase in antibody synthesis compared to control cultures. Delaying cocultivation for 24 hr reduced enhancement to 25%. Thus it appears that an early event—before 24 hr—is involved in CT enhancement. Using ¹²⁵I-CT, it was shown that these effects were not due to CT carry-over. When KLH- and CT-pulsed LNC were cultured in chambers separated by polycarbonate membranes (0.2- to 0.4-μm pore size) antibody production was enhanced 50–80%. Supernates of CT-treated LNC also enhanced antibody production by KLH-treated LNC. These results suggest that CT triggers the release of soluble factor(s) which enhance(s) antibody synthesis by antigen-primed and antigen-challenged LNC.     

A preliminary study on serological assay for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 238 admitted hospital patients

In this study, we aimed to evaluate the diagnostic value of serological assay for SARS-CoV-2. A newly-developed ELISA assay for IgM and IgG antibodies against N protein of SARS-CoV-2 was used to screen the serums of 238 admitted hospital patients between February 6 and February 14, 2020 with confirmed or suspected SARS-CoV-2. SARS-CoV-2 RNA was detected on pharyngeal swab specimens using real time RT-PCR. 194 (81.5%) of the serums were detected to be antibody (IgM and/or IgG) positive, significantly higher than the positive rate of viral RNA (64.3%). There was no difference in the positive rate of antibodies between the confirmed patients (83.0%, 127/153) and the suspected patients (78.8%, 67/85), whose nucleic acid tests were negative. The antibody positive rates were very low in the first five days after initial onset of symptoms, and then rapidly increased as the disease progressed. After 10 days, the antibody positive rates jumped from below 50% to over 80%. However, the positive rates of viral RNA maintained above 60% in the first 11 days after initial onset of symptoms, and then rapidly decreased. Overall, the suspected patients were most likely infected by SARS-CoV-2. Before the 11th day after initial onset of symptoms, nucleic acid test is key for confirmation of viral infection. The combination of serological assay can greatly improve the diagnostic efficacy. After the 11th day post-disease onset, the diagnosis for viral infection should be majorly dependent on serological assay.     

Cytokine gene expression (IL-2 and IL-6) in tumoral lymph node pathology]

Cytokine gene expression could be studied by both immunohistochemistry and in situ hybridization. These techniques allowed us to demonstrate the role of IL-6 and IL-2 in the pathophysiology of Castelman's disease and CD25 positive malignant lymphomas, respectively.     

Amplification of lymph node cell tube leukocyte adherence inhibition (LAI) reactivity by leukocyte adherence inhibition factor (LAIF)

This investigation examines the immunologic basis for specific antigen-induced tube leukocyte adherence inhibition (LAI) reactivity of draining lymph node cells (LNC) from dogs with canine transmissible venereal sarcoma (CTVS). CTVS regressor LNC, macrophage-depleted LNC, and enriched T lymphocyte fractions, but not enriched B lymphocyte fractions, were specifically reactive to CTVS antigen extract in direct tube LAI. In addition, regressor LNC amplified tube LAI responses by generating supernatants with leukocyte adherence inhibition factor (LAIF) activity for normal dog indicator LNC and enriched peripheral blood mononuclear cells (PBMC) in an indirect tube LAI assay. However, macrophage-depleted LNC and enriched T lymphocyte fractions failed to generate supernatants with LAIF activity, suggesting that macrophage accessory cells play a central role in the amplification of tube LAI. Interestingly, CTVS regressor peripheral blood leukocytes (PBL) and PBMC, which were specifically reactive in direct tube LAI, also failed to generate supernatants with LAIF activity. These findings demonstrate a distinction between LAIF-mediated amplification and direct tube LAI reactivity, and suggest that leukocyte populations with differing cellular proportions and from different immunologic compartments may participate in tube LAI via different mechanisms.     

Effect of guinea pig thyroglobulin in incomplete Freund's adjuvant on experimental autoimmune thyroiditis induced by in vitro-activated lymph node cells

Guinea pigs injected with guinea pig thyroglobulin (GPTG) in incomplete Freund's adjuvant (IFA) have been shown to be unresponsive to challenge with GPTG in complete Freund's adjuvant (CFA). However, effector cells which transfer experimental autoimmune thyroiditis (EAT) can be demonstrated in cultured lymph node cells (LNC) of unresponsive animals, indicating that GPTG in IFA does not suppress the initial sensitization of EAT effector cells. LNC from unresponsive animals were unable to suppress the in vitro activation of effector LNC or to suppress EAT when cotransferred with effector cells. When GPTG in IFA was given to animals which were used as recipients of effector cells, the production of EAT was markedly suppressed. These results suggest that GPTG in IFA can suppress EAT either by preventing effector cells from interacting with the thyroid or by interfering with the function of a cell in the normal recipient which may interact with effector cells to result in the lesions of EAT.