Targeting Topics 15q1

The rate of fall of blood glucose determines the necessity of forebrain-projecting catecholaminergic neurons for male rat sympathoadrenal responses.

Jokiaho AJ, Donovan CM, Watts AG.

Diabetes 63(8):2854-2865, 2014.

Different sets of glucosensors detect insulin-induced hypoglycemia depending on the onset rate. This detection controls the activation of sympathoadrenal counterregulatory responses (CRRs). Slow onset hypoglycemia, common with insulin therapy, is detected by glucosensors in the portal-mesenteric veins. Fast onset is detected by brain elements. The authors lesioned hindbrain catecholaminergic neurons to determine which set of responses-they interact with. Rats received 42 ng bilateral injections of Anti-DBH-SAP (Cat. #IT-03) into the paraventricular nucleus of the hypothalamus. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The data indicate that these neurons are critical for detection of slow-onset insulin-induced hypoglycemia.

Intratumoral anti-HuD immunotoxin therapy for small cell lung cancer and neuroblastoma.

Ehrlich D, Wang B, Lu W, Dowling P, Yuan R.

J Hematol Oncol 7(1):91, 2014.

HuD protein is a 40-kDa neuronal RNA-binding protein that is expressed in 100% of small cell lung cancer (SCLC) tumor cells. An anti-HuD monoclonal was biotinylated and combined with Streptavidin-ZAP (Cat. #IT-27); this conjugate was tested both in vitro and in vivo. Anti-HuD-SAP eliminated NCI-H69 and Neuro-2a cells at an EC50 of <0.5 μg/ml. 1 mg/kg of the conjugate injected directly into subcutaneous tumors generated in mice resulted in a temporary lack of tumor growth or regression of the tumor. The results demonstrate the potential of HuD as a therapeutic target for SCLC and neuroblastoma.

Respiratory function after selective respiratory motor neuron death from intrapleural CTB-saporin injections.

Nichols NL, Vinit S, Bauernschmidt L, Mitchell GS.

Exp Neurol 267:18-29, 2015.

Amyotrophic lateral sclerosis (ALS) ultimately causes death from ventilator failure. Genetic models of ALS suffer from high variability of the rate, timing, and extent of respiratory motor neuron death. The authors created a novel model of induced respiratory motor neuron death using CTB-SAP (Cat. #IT-14). Rats received 25 μg or 50 μg intrapleural injections of CTB-SAP; Saporin (Cat. #PR-01) was used as a control. After 7 days, motor neuron survival approximated what is seen in end-stage ALS rats, while there was minimal cell death in other brainstem or spinal cord regions. CTB-SAP also caused microglial activation, decreased breathing during chemoreceptor stimulation, and diminished phrenic motor output in anesthetized rats – all hallmarks of ALS.

Hypocretin/orexin antagonism enhances sleep-related adenosine and GABA neurotransmission in rat basal forebrain.

Vazquez-DeRose J, Schwartz MD, Nguyen AT, Warrier DR, Gulati S, Mathew TK, Neylan TC, Kilduff TS.

Brain Struct Funct 221(2):923-940, 2016.

The basal forebrain (BF) is one of the regions receiving excitatory input from orexin neurons. The authors investigated the hypothesis that orexin antagonists induce sleep at least in part by interfering with the facilitation of BF neurons. Rats received bilateral 500-ng injections of 192-IgG-SAP (Cat. #IT-01) into the BF. Lesioned animals displayed no abnormal responses to a benzodiazepine agonist or vehicle. An orexin antagonist, however, was less effective than the control at inducing sleep in lesioned rats.

Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells.

Sims S, Klenerman P.

Eur J Immunol 45(1):113-118, 2015.

The standard CD8+ T-cell response to infection is a rapid proliferation followed by a reduction in number after the infection is cleared. Murine cytomegalovirus is an exception in that an infection generates a life-long latency with low-level sporadic replication. Immunodominant cells accumulate over time and stabilize at a high frequency. The authors examined a paradoxical boost following depletion of these cells with an M38 antibody attached to Streptavidin-ZAP (Cat. #IT-27). Mice were treated with 44 pM intraperitoneal injections. M38 is an epitope present on the effector CD8+ T cells. Following a significant depletion of cells, the population rebounded and reached a higher percentage of total CD8+ T-cells than before the depletion.

A combination of targeted toxin technology and the piggyBac-mediated gene transfer system enables efficient isolation of stable transfectants in nonhuman mammalian cells.

Sato M, Inada E, Saitoh I, Matsumoto Y, Ohtsuka M, Miura H, Nakamura S, Sakurai T, Watanabe S.

Biotechnol J 10(1):143-153, 2014.

In this work the authors developed a new transfection strategy that takes advantage of the fact that many cell lines endogenously express α-1,3-galactosyltransferase (α-Gal), the target of rIB4-SAP (Cat. #IT-10). After transfection low expressing or non-transfected cells are killed by an application of rIB4-SAP at 80 μg/ml for 2 hours. The surviving cells eventually express α-Gal again, and require no selective agent to maintain expression of the gene of interest. These transfected cells can be transfected again using the same method.

Cholinergic neurons of the basal forebrain mediate biochemical and electrophysiological mechanisms underlying sleep homeostasis.

Kalinchuk AV, Porkka-Heiskanen T, McCarley RW, Basheer R.

Eur J Neurosci 41(2):182-195, 2015.

Previous work has indicated that non-rapid eye movement during recovery sleep after sleep deprivation requires cholinergic neurons in the BF. The authors examined how BF cholinergic neurons affect the levels of HSP markers during sleep deprivation. Rats received 230-ng injections of 192-IgG-SAP (Cat. #IT-01) into the horizontal limb of the diagonal band/substantia innominata/ magnocellular preoptic area. The results indicate that cholinergic neurons in the BF are important for regulating the biochemical and EEG mechanisms that contribute to HSP.

Eye-specific retinogeniculate segregation proceeds normally following disruption of patterned spontaneous retinal activity.

Speer CM, Sun C, Liets LC, Stafford BK, Chapman B, Cheng HJ.

Neural Dev 9(1):25, 2014.

The authors administered 0.88-1.66 μg of an Anti-VaChT-SAP custom conjugate to ferrets with an intraocular injection. Although the lesioned animals demonstrated normal eye-specific retinogeniculate development, there were significant abnormalities in spontaneous retinal activity. These differences in activity manifested themselves as eye-specific segregation defects.

Role of spinal bombesin-responsive neurons in nonhistaminergic itch.

Akiyama T, Tominaga M, Takamori K, Carstens MI, Carstens E.

J Neurophysiol 112(9):2283-2289, 2014.

Recent papers have demonstrated that pruritogen-evoked scratching behavior is reduced or eliminated by intrathecal injection of Bombesin-SAP (Cat. #IT-40). In this work the authors build on those data by investigating if spinal neurons that are responsive to pruritogens administered intradermally are also responsive to a spinal infusion of bombesin. Through the use of intradermal chloroquine injections, spinal superfusion of bombesin, and noxious pinch, the overlap of neurons processing itch and nociception was examined. The results demonstrate that chloroquine- and bombesin-sensitive neurons are involved in the transmission of itch, and that these are a separate neuronal population from those involved in nociception.

Efficient elimination of CD103-expressing cells by anti-CD103 antibody drug conjugates in immunocompetent mice.

Mang Y, Zhao Z, Zeng Z, Wu X, Li Z, Zhang L.

Int Immunopharmacol 24(1):119-127, 2015.

Previous work has demonstrated that an M290-SAP custom conjugate promoted the long-term survival of pancreatic islet allografts by reducing the number of CD103+ cells. M290 is an antibody that targets CD103. Systemic use of the saporin conjugate can result in toxicity and bystander effects to the animal. In this work the authors used M290 conjugated to three different cytotoxic agents in order to avoid these bystander effects. The various reagents were compared in several assays, including internalization studies, flow cytometry, and cytotoxicity studies. The results indicate that the alternative cytotoxic drugs can be used systemically with M290 to eliminate CD103+ cells.

Improvements in memory after medial septum stimulation are associated with changes in hippocampal cholinergic activity and neurogenesis.

Jeong da U, Lee JE, Lee SE, Chang WS, Kim SJ, Chang JW.

Biomed Res Int 2014:568587, 2014.

Deep brain stimulation (DBS) is a technique by which electrical impulses are applied to specific areas of the brain as therapy for various disorders. In this work the authors examined the mechanisms by which DBS can treat dementia. Rats received 5.04 μg intracerebroventricular injections of 192-IgG-SAP (Cat. #IT-01); some rats also received an electrode implanted into the medial septum. Lesioned animals displayed deficits in water maze testing – this deficit was eliminated for the group that received electrical stimulation to the medial septum. The stimulated group also displayed an increase in hippocampal cholinergic activity as well as neurogenesis, indicating that DBS has therapeutic potential.

NK1-receptor-expressing paraventricular nucleus neurones modulate daily variation in heart rate and stress-induced changes in heart rate variability.

Feetham CH, Barrett-Jolley R.

PHY2 2(12):e12207, 2014.

Neurons in the paraventricular nucleus (PVN) project to the medulla and spinal cord, regulating heart rate and blood pressure. Although the activity of these neurons becomes elevated during heart failure, their role in overall cardiovascular control is unclear. The authors lesioned the PVN of rats with 2 ng injections of SSP-SAP (Cat. #IT-11). Heart rate variability during the experiment was measured using a high/low frequency ratio in response to psychological stress. The variability response of lesioned rats was lower than that of controls, and a shift in daily heart rate variation was seen as well. The authors conclude that neurokinin-1 expressing neurons in the PVN couple the cardiovascular system to the daily heart rate as well as the sympathetic response to psychological stress.

Targeted toxin-based selectable drug-free enrichment of Mammalian cells with high transgene expression.

Sato M, Akasaka E, Saitoh I, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S.

Biology (Basel) 2(1):341-355, 2013.

Cell transfection is a powerful tool for evaluation of function and expression of newly discovered genes as well as for both small and large scale eukaryotic expression of proteins. Most transfection strategies require a selection agent to eliminate cells that do not internalize the plasmid containing the gene of interest. Subsequent maintenance of the tranfected cells requires the presence of the selection agent, and the expression levels of the gene of interest have to be evaluated on a cell by cell basis. In this work the authors designed a system utilizing 50 μg/ml rIB4-SAP (Cat. #IT-10) to eliminate non-transfected cells and select for strong expression of the gene of interest. The data demonstrate that this technique will generate stable transfected cells that express the gene of interest at high levels.