Comparison of mouse strains using the local lymph node assay

The local lymph node assay (LLNA), as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), only allows for the use of CBA mice. The objective of these studies was to begin to assess the response of chemical sensitizers in the LLNA across six strains of female mice (C57BL/6, SJL/J, BALB/c, B6C3F1, DBA/2 and CBA). The moderate sensitizer alpha-hexylcinnamaldehyde (HCA) was chosen as the test chemical, while toluene diisocyanate (TDI) and 2,4-dinitrofluorobenzene (DNFB) were evaluated at single concentrations as positive controls. Draining lymph node cell proliferation following acetone exposure varied across strains. SJL mice had a significantly higher degree of proliferation with 2111 d.p.m./2 nodes. The remaining five strains demonstrated responses which ranged from 345 to 887 dpm/2 nodes. DBA/2, B6C3F1, BALB/c and CBA mice had essentially equal levels of lymph node proliferation following exposure to the three chemicals. While C57BL/6 mice gave similar results as CBA mice following DNFB and HCA administration, the LLNA response to TDI was considerably lower. SJL mice provided low stimulation indexes (SI) values for all three chemicals evaluated. Regardless of the level of LLNA response, all six mouse strains identified the sensitization potential of HCA, TDI or DNFB. Based on these studies, DBA/2, B6C3F1 and BALB/c mice are good choices for continued evaluation as additional mouse strains for use in the LLNA.     

Clinical trial billing compliance at academic medical centers.

Academic medical centers (AMCs) face a weighty regulatory compliance risk in billing Medicare and other third-party payors for clinical trial services. The culture and structure of many AMCs present significant challenges when attempting to coordinate the necessary information and operations to determine whether clinical trial services can be billed to payors or should be charged to an internal research account that tracks the sponsorship monies or grant award. The federal rules are complex, but at base they revolve around the principle that payors should not be billed for clinical trial services that are either being paid by someone else or not provided for the direct clinical care of the patient. With proper coordination and leadership, an AMC can coordinate its efforts, transform its decentralized culture, and achieve a compliance confidence level. Beginning in 2003, the authors helped guide Rush University Medical Center through its landmark voluntary disclosure and settlement with the United States on cancer clinical trial billing. The corrective actions adopted by Rush have set a model that can be adapted for other AMCs. The attention to Medicare's clinical trials coverage rules brought about by the Rush settlement has led to the Medicare program revisiting its rules and proposing a number of changes that help clarify Medicare policy.     

Immune serum from mice contact-sensitized with picryl chloride contains an antigen-specific T cell factor that transfers immediate cutaneous reactivity

This report describes an activity in serum from mice that were contact-sensitized with picryl chloride (PCl) 1 to 4 days earlier. Immune serum, when given i.v., transfers the ability to elicit an immediate hypersensitivity-like ear swelling reaction in naive recipients following local challenge with PCl. This serum activity is due to an antigen-binding T cell factor that shares some properties with IgE antibody. The activity is antigen specific, and due to an antigen-binding moiety that is heat labile (56 degrees C, 4 h). However, unlike IgE antibody the serum activity is resistant to reduction and alkylation, and is retained by columns of Sepharose beads coupled with polyclonal or monoclonal antibodies that react with antigen-specific T cell factors from other systems. These columns did not retain IgE antibody activity in our experiments. Importantly, the serum activity was not retained by columns linked with antibodies directed to mouse immunoglobulins, which do retain IgE activity. We conclude from these data that the activity in PCl immune serum is not caused by IgE antibody, and is due to the presence of the previously described antigen-specific T cell factor (PCl-factor), that can activate serotonin-containing cells, such as mast cells, to release the vasoactive amine serotonin. PCl-factor transfers the ability to elicit an immediate hypersensitivity-like reaction that is an early component of delayed-type hypersensitivity. The presence of this T cell factor in the serum of actively sensitized mice provides a means to sensitize tissues throughout the body for this required, initial, serotonin-dependent component of delayed-type hypersensitivity reactions.     

Mice bearing the ob/ob mutation have impaired immunity.

The obese mutant mouse C57BL/6J ob/ob showed impaired ability to reject skin grafts or react to a contact-sensitising agent in comparison with littermate controls (either +/ob or +/+). Ability of spleen cells from mice bearing the ob/ob mutation to produce a graft-versus-host reaction in C57BL/6J X DBA/2J F1 hybrid mice was not impaired.     

Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice

The choice of the type of instrument to measure delayed-type hypersensitivity (DTH) in mice, as assayed by ear swelling reactions, influences the experimental results. When a caliper that applies little pressure to the ears is employed, DTH reactions in ears of mice sensitized to picryl chloride show an early onset at 2 h after challenge, comparable swelling at 4 h and a slow rise to a 24 h classical peak response thereafter. In contrast, 3 different micrometers that apply more pressure to the ears reveal a biphasic pattern of ear swelling reactions in mice immunized and challenged with picryl chloride. The early component of DTH measured by these micrometers peaks 2 h after challenge. Thereafter the measured ear thickness declines, and the onset of the classical delayed reaction is detected at 12 h after ear challenge. Yet another instrument, that in contrast to the caliper and micrometers mentioned above, applies all the pressure to only a very restricted area of the ear, fails to detect an early sweeling reaction; the delayed reaction is first detected at 12 h after ear challenge and rises there after to a 24 h peak.The differences in outcome of the assays using the different instruments indicate that the early component or DTH reactions differs from the late component of DTH reactions in that the early swelling is easier to compress when pressure is applied by the instrument used for measurement. This is probably caused by the fact that the late reactions are due to a cellular infiltrate, whereas the early reactions are edematous in character, and are due to accumulation of plasma components.     

Lewisham bereavement project: death and the life that's left

Coping with bereaved relatives is a problem, both emotional and practical, for nursing staff. At Lewisham Hospital a scheme was set up bringing in the existing volunteer system to support the bereaved. Here, Janet Keyte, sector administrator; Bette Meade, voluntary services organiser; and Philip Nye, senior nursing officer, give the history of the Lewisham Volunteer Bereavement Project.     

Antitransferrin receptor antibody-RNase fusion protein expressed in the mammary gland of transgenic mice

The CC chemokine eotaxin is potent eosinophil-selective chemoattractant, and it is thought that the function of eotaxin is closely related to the recruitment of eosinophils in certain inflammatory reactions. In order to learn more about the biological role of this molecule, we have developed a new sandwich ELISA method to measure human eotaxin using two monoclonal anitibodies and purified recombinant eotaxin as a standard. The minimal detectable concentration of eotaxin in this assay was 1.5 pg/ml, and the working range was 3.1--200 pg/ml with low CVs (< 10%). Both within- and between-run precision levels were less than 6.7% of the CVs. The dilution curves of two serum and two spiked plasma samples showed good linearity and the recovery range was 92.8--103.3%. No cross-reactivity was found with other similar chemokines. MCP-1, MCP-2, MCP-3, MCP-4, eotaxin-2 and RANTES. This assay was sensitive enough to measure the circulating eotaxin levels of healthy volunteers. However, the eotaxin levels in serum samples (mean+/-SD; 68.6+/-13.4 pg/ml, n=15) were significantly higher than those in matched plasma samples (19.2+/-5.4 pg/ml) separated from blood collected in tubes containing EDTA. Kinetic studies revealed that the eotaxin levels in serum markedly increased depending on the elapsed time before separation from blood cells, but such changes in EDTA-plasma were negligible up to 4 h at 25 degrees C. Our new ELISA is an accurate and useful method for quantifying human eotaxin in blood and demonstrates that the process of preparing blood samples affects the measurement of the eotaxin levels.     

The tyrosine kinase Lyn is required for B cell development beyond the T1 stage in the spleen: rescue by over-expression of Bcl-2

We have analyzed the effects of deficiency in the tyrosine kinase Lyn on B cell development using transgenic mice that express a B cell antigen receptor (BCR) of defined specificity (3-83,anti-H-2K(k or b)). In the absence of Lyn, immature B cells are abundant in the bone marrow and spleen up until the T1 stage (IgM(hi) IgD(-) CD21(-)CD23(-)), after which B cell development is severely impaired. The small number of mature B cells that do develop in Lyn-deficient mice express normal levels of the transgenic BCR and lack expression of CD80 and CD86, suggesting they are not activated. In Lyn-deficient animals the presence of a Bcl-2 transgene leads to a dramatic increase in B cell numbers and restores T2 stage (IgM(hi) IgD(hi) CD21(hi) CD23(int)) and mature populations. In 3-83 lyn-/- Bcl-2 Tg mice, a population of lambda-positive cells that also express the 383 idiotype is evident, suggesting that in the absence of lyn isotype exclusion by the transgenic BCR is less efficient. The results indicate that Lyn plays a positive role in the selection and survival of mature B cells in addition to its previously documented negative role in tolerance and B cell activation.