SUMMARY Effector memory T (Tem) cells are essential mediators of autoimmune disease and delayed-type hypersensitivity (DTH), a convenient model for two-photon imaging of Tem cell participation in an inflammatory response. Shortly (3 hr) after entry into antigen-primed ear tissue, Tem cells stably attached to antigen-bearing antigen-presenting cells (APCs). After 24 hr, enlarged Tem cells were highly motile along collagen fibers and continued to migrate rapidly for 18 hr. Tem cells rely on voltage-gated Kv1.3 potassium channels to regulate calcium signaling. ShK-186, a specific Kv1.3 blocker, inhibited DTH and suppressed Tem cell enlargement and motility in inflamed tissue but had no effect on homing to or motility in lymph nodes of naive and central memory T (Tcm) cells. ShK-186 effectively treated disease in a rat model of multiple sclerosis. These results demonstrate a requirement for Kv1.3 channels in Tem cells during an inflammatory immune response in peripheral tissues. Targeting Kv1.3 allows for effector memory responses to be suppressed while central memory responses remain intact.

Kv1.3 potassium channels maintain the membrane potential of effector memory (T EM ) T cells that are important mediators of multiple sclerosis, type 1 diabetes mellitus, and rheumatoid arthritis. The polypeptide ShK-170 (ShK-L5), containing an N-terminal phosphotyrosine extension of the Stichodactyla helianthus ShK toxin, is a potent and selective blocker of these channels. However, a stability study of ShK-170 showed minor pH-related hydrolysis and oxidation byproducts that were exacerbated by increasing temperatures. We therefore engineered a series of analogs to minimize the formation of these byproducts. The analog with the greatest stability, ShK-192, contains a nonhydrolyzable phosphotyrosine surrogate, a methionine isostere, and a C-terminal amide. ShK-192 shows the same overall fold as ShK, and there is no evidence of any interaction between the N-terminal adduct and the rest of the peptide. The docking configuration of shows the N-terminal para-phosphonophenylalanine group lying at the junction of two channel monomers to form a salt bridge with Lys 411 of the channel. ShK-192 blocks Kv1.3 with an IC 50 of 140 pM and exhibits greater than 100-fold selectivity over closely related channels. After a single subcutaneous injection of 100 g/kg, ϳ100 to 200 pM concentrations of active peptide is detectable in the blood of Lewis rats 24, 48, and 72 h after the injection. ShK-192 effectively inhibits the proliferation of T EM cells and suppresses delayed type hypersensitivity when administered at 10 or 100 g/kg by subcutaneous injection once daily. ShK-192 has potential as a therapeutic for autoimmune diseases mediated by T EM cells.

BackgroundNuclear receptor subfamily 1, group I, member 2 (NR1I2), commonly known as steroid and xenobiotic receptor (SXR) in humans, is a key ligand-dependent transcription factor responsible for the regulation of xenobiotic, steroid, and bile acid metabolism. The ligand-binding domain is principally responsible for species-specific activation of NR1I2 in response to xenobiotic exposure.ObjectivesOur objective in this study was to create a common framework for screening NR1I2 orthologs from a variety of model species against environmentally relevant xenobiotics and to evaluate the results in light of using these species as predictors of xenobiotic disposition and for assessment of environmental health risk.MethodsSixteen chimeric fusion plasmid vectors expressing the Gal4 DNA-binding domain and species-specific NR1I2 ligand-binding domain were screened for activation against a spectrum of 27 xenobiotic compounds using a standardized cotransfection receptor activation assay.ResultsNR1I2 orthologs were activated by various ligands in a dose-dependent manner. Closely related species show broadly similar patterns of activation; however, considerable variation to individual compounds exists, even among species varying in only a few amino acid residues.ConclusionsInterspecies variation in NR1I2 activation by various ligands can be screened through the use of in vitro NR1I2 activation assays and should be taken into account when choosing appropriate animal models for assessing environmental health risk.

Drawing blood from rodents is necessary for a large number of both in vitro and in vivo studies. Sites of blood draws are numerous in rodents: retro-orbital sinus, jugular vein, maxillary vein, saphenous vein, heart. Each technique has its advantages and disadvantages, and some are not approved any more in some countries (e.g., retro-orbital draws in Holland). A discussion of different techniques for drawing blood are available 1-3 . Here, we present two techniques for drawing blood from rats, each with its specific applications.Blood draw from the saphenous vein, provided it is done properly, induces minimal distress in animals and does not require anesthesia. This technique allows repeated draws of small amounts of blood, such as needed for pharmacokinetic studies 4,5 , determining plasma chemistry, or blood counts 6 .Cardiac puncture allows the collection of large amounts of blood from a single animal (up to 10 ml of blood can be drawn from a 150 g rat). This technique is therefore very useful as a terminal procedure when drawing blood from the saphenous would not provide a large enough sample. We use cardiac puncture when we need sufficient amounts of serum from a specific strain of rats to grow T lymphocyte lines in vitro 4-9 .

Purpose [18F]Mefway is a novel radiotracer specific to the serotonin 5-HT1A receptor class. In preparation for using this tracer in humans, we have performed whole-body PET studies in mice to evaluate the biodistribution and dosimetry of [18F]Mefway. Methods Six mice (three females and three males) received IV injections of [18F]Mefway and were scanned for 2 h in an Inveon-dedicated PET scanner. Each animal also received a high-resolution CT scan using an Inveon CT. The CT images were used to draw volume of interest on the following organs: the brain, large intestine, stomach, heart, kidneys, liver, lungs, pancreas, bone, spleen, testes, thymus, gallbladder, uterus, and urinary bladder. All organ time-activity curves without decay correction were normalized to the injected activity. The area under the normalized curves was then used to compute the residence times in each organ. Data were analyzed using PMOD and Matlab software. The absorbed doses in mouse organs were computed using the RAdiation Dose Assessment Resource animal models for dose assessment. The residence times in mouse organs were converted to human values using scale factors based on differences between organ and body weights. OLINDA/EXM 1.1 software was used to compute the absorbed human doses in multiple organs for both female and male phantoms. Results The highest mouse residence times were found in the liver, urinary bladder, and kidneys. The largest doses in mice were found in the urinary bladder (critical organ), kidney, and liver for both females and males, indicating primary elimination via urinary system. The projected human effective doses were 1.21E–02 mSv/MBq for the adult female model and 1.13E–02 mSv/MBq for the adult male model. The estimated human biodistribution of [18F]Mefway was similar to that of [11C]WAY 100,635, a 5-HT1A tracer for which dosimetry has been evaluated in humans. Conclusions The elimination of radiotracer was primarily via the kidney and urinary bladder with the urinary bladder being the critical organ. Whole-body mouse imaging can be used as a preclinical tool to provide initial estimates of the absorbed doses of [18F]Mefway in humans.