custom-conjugates

Porter LM, Alenciks E, Frazier K, Porter A, Fraley GS (2015) Lack of effects on growth and body weight gain after elimination of the leptin receptor from the brain of immature Pekin drakes. Neuroscience 2015 Abstracts 613.04/R19. Society for Neuroscience, Chicago IL.

Summary: The presence of the hormone leptin (LEP) is a controversial topic in the field of avian physiology. While LEP is well understood in mammals, the hormone has not been definitively verified in avian species. Although the hormone remains elusive, the leptin receptor (LEPR) has been identified and sequenced in multiple avian species. Its role, however, remains unclear. To attempt to deduce the role of the leptin system in birds, we focused on altering the leptin receptor expression in the brain of immature Pekin ducks. We hypothesized that eliminating the LEPR-expressing neurons of the hypothalamus would elicit an increase in body weight, as is the case for mammals. To test this hypothesis, we injected stereotaxically 3 ul of a solution containing a monoclonal antibody (anti-LEPR) conjugated to saporin (LSAP, 100 ng/ul) was injected into the lateral ventricle of 10 day old Pekin ducks (LSAP, N = 10). Control group animals (SAP) were injected with unconjugated antibody and saporin at equimolar concentrations to the LSAP. Ducks were weighed weekly starting at 3 days of age. After a final weight was obtained at 50 days of age, ducks were euthanized and a blood sample was collected and sent out for an avian panel to assay serum glucose and free fatty acids. We found that the elimination of LEPR had no significant effect on the body weights of the ducks (p>0.05). In addition, The CBC panel did not reveal any significant differences in the overall health of the ducks in each treatment group. Our data indicates LEPR may not play a significant role in the regulation of body weight or growth in juvenile ducks.

Related Products: Custom Conjugates, Saporin (Cat. #PR-01)

Wiley RG (2015) Nociceptive effects of neurotensin(NTS)- and somatostatin(SST)-toxin conjugates applied to the lumbar dorsal horn in rats. Neuroscience 2015 Abstracts 418.11/O12. Society for Neuroscience, Chicago IL.

Summary: Intrathecal injections of NTS or SST have been reported to be anti-nociceptive, and in the case of SST, analgesic in humans. Preliminary experiments in our lab previously showed that lumbar intrathecal injection of the excitatory neuropeptide, NTS, or the inhibitory neuropeptide, SST, conjugated to the ribosome inactivating protein, saporin (sap), produced compulsive scratching/biting of hindquarters resulting in loss of fur and skin. This was thought likely due to pain and/or itching from selective loss of superficial dorsal horn nociceptive inhibitory interneurons expressing NTS receptors. Subsequent experiments using lumbar intrathecal injections of NTS-cholera toxin A chain conjugate resulted in prolonged anti-nociception on hotplate, tail flick and von Frey testing, that was not reversed by naloxone and lasted several days, likely due to sustained activation of the same neurons. The present study sought to determine if the lesions produced by NTS-sap or SST-sap alter nociceptive responses. In the present study, rats, under isoflurane anesthesia, were injected intrathecally using temporarily-placed subarachnoid catheters over the lumbar enlargement with 10 ul of sterile preservative-free normal saline containing either 300-400 ng of NTS-sap, 1 ug of SST-sap or 1 ug blank-sap (control) from Advanced Targeting Systems, San Diego, CA. Catheters were flushed with an additional 10 ul of saline. After post-surgical recovery, the rats were then observed for scratching/biting their hindquarters, nocifensive responses on the hotplate, von Frey mechanical probing of the hindpaws, and on operant thermal escape. 4 of 11 NTS-saporin rats and 5 of 9 SST-saporin rats, but none of 9 blank-saporin rats began scratching within 8-47 days after toxin conjugate injection. Hotplate nocifensive reflex testing at 44.5°C and 47°C showed no significant difference between the groups. Von Frey, operant thermal escape testing and anatomic studies are in progress to further specify the functional effects of the toxin conjugate injections and to identify the dorsal horn neurons being destroyed. The results to date are interpreted as consistent with a possibly unique role for NTS and/or SST receptor-expressing superficial dorsal horn inhibitory interneurons in nociception and/or itch. Excitatory/activating moieties such as cholera toxin A subunit targeted by conjugation to NTS or SST may offer a novel approach to enhance inhibition in nociceptive dorsal horn neurons and to produce analgesia by a non-opioid mechanism.

Related Products: Neurotensin-CTA (Cat. #IT-60), Neurotensin-SAP (Cat. #IT-56), Blank-SAP (Cat. #IT-21), Custom Conjugates

Garcia-Castillo M, Tran T, Bobard A, Renard H, Rathjen S, Dransart E, Stechmann B, Lamaze C, Lord M, Cintrat J, Enninga J, Tartour E, Johannes L (2015) Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin. J Cell Sci 128:2373-2387. doi: 10.1242/jcs.169383

Summary: Bacterial and plant toxins rely on various trafficking pathways to reach intracellular targets. Shiga and Shiga-like toxins have been found to be moved via vesicular transport through several subcellular structures on the way to the cytosol. Shiga toxin (STx) is the cause of hemolytic uremic syndrome, for which there is no effective treatment. In order to better understand the mechanisms of STx membrane translocation the authors used a custom conjugate of the receptor-binding B-subunit of STx (STxB) and saporin (Custom conjugation provided by Advanced Targeting Systems). In vitro assays demonstrated that STxB-SAP did not use retrograde transport to the Golgi complex in order to reach the cytosol. This information has relevance to antigen cross-presentation of antigen-presenting cells.

Related Products: Custom Conjugates

Bostad M, Olsen C, Peng Q, Berg K, Høgset A, Selbo P (2015) Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization – A minimally invasive cancer stem cell-targeting strategy. J Control Release 206:37-48. doi: 10.1016/j.jconrel.2015.03.008

Summary: Previously the authors demonstrated the use of photochemical internalization of a custom conjugate consisting of a CD133 antibody coupled to saporin (ATS Custom conjugation). Several cancer cell lines were plated, and incubated in the presence of a photosensitizer with either CD133-SAP at 8.6 pM or Saporin (Cat. #PR-01) at 24 pM. The different concentrations equalized the number of saporin molecules in each sample. A light source was used to initiate the internalization of the molecules. The results indicate that this is a viable strategy for the targeted treatment of cancer stem cells.

Related Products: Anti-CD133-SAP (Cat. #IT-82), Saporin (Cat. #PR-01), Custom Conjugates

Konsman J, Chaskiel L, Bristow A, Dantzer R (2014) Interleukin-1 receptor-expressing cells in the arcuate hypothalamus mediate peripheral interleukin-1-induced hypophagia. Neuroscience 2014 Abstracts 453.13. Society for Neuroscience, Washington, DC.

Summary: Although the reduction in food intake observed in acute infectious and inflammatory diseases has been proposed to represent a regulated adaptive response, the underlying mechanisms remain incompletely understood. Our previous work has shown that the pro-inflammatory cytokine interleukin-1 (IL-1) can act in the brain to alter behavior during peripheral inflammation. The arcuate nucleus of the rat hypothalamus plays a pivotal role in the regulation of food intake and expresses the signaling interleukin-1 receptor (IL-1R1) (Ericsson et al., J. Comp. Neurol., 1995). However, lesioning of the neuropeptide Y(NPY)- and proopiomelancortin(POMC)-expressing neurons, the two major neuronal populations in the arcuate nucleus regulating food intake, does not attenuate the reduction of food intake after peripheral interleukin-1 administration (Reyes & Sawchenko, J. Neurosci., 2002). Besides neurons, venules and glia constitute the main nervous cell types expressing the signaling interleukin-1 receptor. Moreover, glial cells, and in particular tanycytes in the arcuate nucleus, have been proposed to play a role in the regulation of food intake (Bolborea & Dale, Trends Neurosci., 2013). In the present work, we set out ) to determine if IL1-R1-expressing cells in the hypothalamus mediate reduced food intake in response to peripheral IL-1 administration, and 2) if so, to identify the cell types involved. Cells expressing IL-1R1 were killed by infusion of IL-1 coupled to the intracellular toxin saporin (IL-1-SAP) into the arcuate hypothalamus. Control infusions consisted of uncoupled IL-1 and saporin and PBS. At least one week later rats were injected intraperitoneally with IL1. Intra-arcuate IL-1-SAP attenuated the reduction in food intake after peripheral administration of IL-1, indicating that arcuate cells mediate IL-1-induced hypophagia. Post mortem histochemical analyses of brain sections of the same animals revealed that intra-arcuate IL-1-SAP reduced the number of NPY-neurons, without affecting the number of POMC-neurons or the surface covered by tanycytes. Taken together, these findings indicate that IL-1R-bearing NPY neurons in the arcuate nucleus take part in the reduction of food intake after peripheral IL-1 administration and suggest that hypophagia observed in infectious and inflammatory diseases reflects, at least in part, a regulated response.

Related Products: Custom Conjugates

Mang Y, Zhao Z, Zeng Z, Wu X, Li Z, Zhang L (2015) Efficient elimination of CD103-expressing cells by anti-CD103 antibody drug conjugates in immunocompetent mice. Int Immunopharmacol 24:119-127. doi: 10.1016/j.intimp.2014.11.004

Summary: 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.

Related Products: Custom Conjugates

Speer C, Sun C, Liets L, Stafford B, Chapman B, Cheng H (2014) Eye-specific retinogeniculate segregation proceeds normally following disruption of patterned spontaneous retinal activity. Neural Dev 9:25. doi: 10.1186/1749-8104-9-25

Summary: 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.

Related Products: Custom Conjugates

Ogawa S, Nathan FM, Parhar IS (2014) Habenular kisspeptin modulates fear in the zebrafish. Proc Natl Acad Sci U S A 111(10):3841-3846. doi: 10.1073/pnas.1314184111 PMID: 24567386

Summary: The peptide kisspeptin can be found in several areas of the brain, but its role in regions other than the hypothalamus has not been studied. Zebrafish express kiss1 mRNA which is a conserved ortholog of the mammalian KISSI/KissI making zebrafish a viable model for investigating the role of kisspeptin in various brain systems. Animals received 1 μg of the custom conjugate kiss-SAP (see NK3-SAP, Cat. #IT-63) via an intracranial injection. Blank-SAP (Cat. #IT-21) was used as a control. Reducing Kiss1 immunoreactivity in the habenula and the raphe reduced an invoked fear response, indicating a role for kisspeptin in fear inhibition.

Related Products: NKB-SAP (Cat. #IT-63), Blank-SAP (Cat. #IT-21), Kisspeptin-SAP (Cat. #IT-102)

Quadros EV, Nakayama Y, Sequeira JM (2013) Saporin conjugated monoclonal antibody to the transcobalamin receptor TCblR/320 is effective in targeting and destroying cancer cells. J Cancer Ther 4(6):1074-1081. doi: 10.4236/jct.2013.46122

Summary: Although the transcobalamin receptor (TCb1R) is expressed in most cell types, the expression levels are increased in actively proliferating cells, and decreased in quiescent cells. This expression profile makes the TCb1R an attractive target for cancer therapy. The authors used a custom conjugate of antibodies generated against the TCb1R and saporin to eliminate cancer cell lines in culture. When applying the conjugate to cells in a dosing range of 0.156-5 nM, 2.5 nM was found to have the optimal effect. Given that the level of toxicity was determined by the level of TCb1R expression, targeting the TCb1R may have potential as part of a cancer treatment strategy.

Related Products: Custom Conjugates

Mishra SK, Hoon MA (2013) The cells and circuitry for itch responses in mice. Science 340(6135):968-971. doi: 10.1126/science.1233765

Summary: Although previous work implicated neurons expressing the GRP (gastrin-releasing peptide) receptor were in the pruritic, or itch pathway, transgenic mice lacking natriuretic polypeptide b (Nppb) were almost completely insensitive to itch. Using the custom conjugate Nppb-SAP (Cat. #IT-69), the authors eliminated itch in response to a wide range of pruritic substances in normal mice through the administration of 5 μg of conjugate into the intrathecal space. Even after this lesion, the scratching response to intrathecal GRP was not changed, indicating that the role of GRP is at a later stage than previously hypothesized.

Related Products: Nppb-SAP (Cat. #IT-69)