Goodman RL, Lopez JA, Bedenbaugh MN, Connors JM, Hardy SL< Hileman SM, Coolen LM, Lehman MN (2018) Evidence that the LH surge in ewes involves both neurokinin B-dependent and -independent actions of kisspeptin. Neuroscience 2018 Abstracts 773.20 / YY14. Society for Neuroscience, San Diego, CA.
Summary: It is generally recognized that kisspeptin plays a key role in induction of the LH surge in sheep and we have reported evidence that neurokinin B (NKB) does so as well. Specifically, disrupting NKB signaling in the retrochiasmatic area (RCh) using either an antagonist to its receptor, NK3R, or lesions of NK3R-containing neurons in the RCh with a saporin conjugate (NK3-SAP) reduced the amplitude of the estrogen-induced LH surge by 50%. Because a KISS1R antagonist (p271) also produced a 50% decrease in surge amplitude, we hypothesized that these two systems are organized in series with NKB actions in the RCh stimulating kisspeptin release. If this is the case, then the combination of NK3R lesions and a KISS1R antagonist should produce the same inhibition as either treatment alone. This experiment tested this prediction using a 2 x 2 design. Breeding season ewes were ovariectomized and immediately given an estradiol (E) implant sc and two progesterone implants (CIDRs) intravaginally that produced luteal phase levels of these steroids. Ewes then received bilateral injections of either NK3-SAP (n=6) or Blank-SAP (n=5) into the RCh. Three weeks later, an artificial follicular phase was produced by inserting four 3 cm long E implants 24 hrs after CIDR removal and either saline or p271 was infused into the lateral ventricle for 16-24 hrs after E implantation; LH was monitored every 2-4 hrs for two days. CIDRs were then reinserted and the protocol repeated in a cross-over design so that all ewes received saline and p271 treatment. In Blank-SAP ewes, p271 decreased the peak of the LH surge from 61.2 ± 7.6 to 27.4 ± 4.6 ng/mL and delayed it 8 hrs (from 26.5 ± 0.5 to 34.1 ± 1.2 hrs post E implantation). The NK3-SAP injections alone decreased the peak of the LH surge to 29.7 ± 10.7 ng/mL compared to Blank-SAP, but the peak was not further inhibited by p271 in these NK3-SAP-treated ewes (24.4 ± 1.4 ng/mL). However, p271 delayed the peak of the LH surge (from 28.8 ± 1.2 to 34.8 ± 2.1 hrs post E implantation) in the ewes injected with NK3-SAP. Based on these results, we propose that kisspeptin has two roles in the LH surge in ewes: it initiates the surge independent of NKB signaling in the RCh, and maintains LH secretion during the surge by a NKB-dependent system.
Cua S, Tan HL, Fong WJ, Chin A, Lau A, Ding V, Song Z, Yang Y, Choo A (2018) Targeting of embryonic annexin A2 expressed on ovarian and breast cancer by the novel monoclonal antibody 2448. Oncotarget 9:13206-13221. doi: 10.18632/oncotarget.24152
Objective: To develop mAbs to potentially target oncofetal antigens and be repurposed for antibody or antibody drug conjugate (ADC) therapy.
Summary: The novel IgG1, 2448, was shown to target a unique glycosylated surface epitope on ANXA2. As a possible therapeutic candidate for ovarian and breast cancer, 2448 demonstrated anti-tumor activity via two independent mechanisms of action.
Usage: Cells were seeded in 96-well plates at 1000 or 2000 cells/well. Primary antibody, 2448 or ch2448 (10 μg/mL) was pre-mixed with appropriate secondary saporin conjugate, Mab-ZAP or Hum-ZAP. The most significant decreases in cell viability (20% to 60%) were observed against the epithelial IGROV1 and MCF7 cell lines. ATS created a Custom ADC by direct conjugation of saporin to ch2448 (ch2448-SAP). As a control, an isotype chimeric IgG was also conjugated to saporin (IgG-SAP). Compared to using secondary saporin conjugates, ch2448-SAP induced and increase of 20–30% cytotoxicity.)
Schiroli G, Ferrari S, Conway A, Jacob A, Capo V, Albano L, Plati T, Castiello M, Sanvito F, Gennery A, Bovolenta C, Palchaudhuri R, Scadden D, Holmes M, Villa A, Sitia G, Lombardo A, Genovese P, Naldini L (2017) Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1. Sci Transl Med 9(411):eaan0820. doi: 10.1126/scitranslmed.aan0820
Objective: To study potential approaches to gene therapy in mouse models of severe combined immunodeficiency.
Summary: The threshold of IL2RG gene editing can be reached for safe and efficient correction of SCID-X1 established in a preclinical model in human long-term repopulating HSPCs.
Usage: Biotinylated Anti-CD45 was mixed equimolar to Streptavidin-ZAP and administered as a single dose which caused substantial depletion (~70%) of the HSPC compartments and milder depletion of the more mature cell populations.
Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells.
O’Sullivan J, Carroll D, Cao Y, Salicru A, Bochner B (2018) Leveraging Siglec-8 endocytic mechanisms to kill human eosinophils and malignant mast cells. J Allergy Clin Immunol 141:1774-1785.e1777. doi: 10.1016/j.jaci.2017.06.028
Summary: Therapeutic payloads can be targeted selectively to eosinophils and malignant mast cells by exploiting this Siglec-8 endocytic pathway.
Usage: Eosinophil cell death was assessed with 2C4 mAb or isotype control (both at 2.5 μg/mL).Related Products: Custom Conjugates
Sedlik C, Heitzmann A, Viel S, Ait Sarkouh R, Batisse C, Schmidt F, De La Rochere P, Amzallag N, Osinaga E, Oppezzo P, Pritsch O, Sastre-Garau X, Hubert P, Amigorena S, Piaggio E (2016) Effective antitumor therapy based on a novel antibody-drug conjugate targeting the Tn carbohydrate antigen. Oncoimmunology 5:e1171434. doi: 10.1080/2162402X.2016.1171434
Summary: Scientists wanted to study the potential of Chi-Tn, a monoclonal antibody against a glycol-peptidic tumor-associated antigen, as an anticancer antibody-drug conjugate. They demonstrated that Chi-Tn specifically targeted tumor cells in vivo, using flow cytometry and deconvolution microscopy to show that Chi-Tn is rapidly internalized. Chi-Tn-SAP (ATS Custom Services) effectively killed Tn-positive cells, but had no effect on Tn-negative cells. Saporin (Cat. #PR-01) was used as control. The cytotoxicity of the Chi-Tn-SAP correlated with the level of tumoral Tn expression.
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.
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.
Retrograde transport is not required for cytosolic translocation of the B-subunit of Shiga toxin.
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.
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.
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.
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.
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.
Habenular kisspeptin modulates fear in the zebrafish.
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
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.
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.
The cells and circuitry for itch responses in mice.
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.
Kato J, O’Donnell RT, Abuhay M, Tuscano JM (2012) Efficacy and toxicity of a CD22-targeted antibody-saporin conjugate in a xenograft model of non-Hodgkin’s lymphoma. Oncoimmunology 1(9):1469-1475. doi: 10.4161/onci.21815
Summary: CD22 is a B-cell-specific antigen found on many B-cell malignancies. It is not expressed by stem cell precursors, and is rapidly internalized when bound by an antibody. In this work, the authors use a custom conjugate of anti-CD22 (mAb HB22.7) and saporin in a cytotoxicity assay on non-Hodgkin’s lymphoma cell lines, as well as in a mouse tumor model. The dosing for the tumor model was 1 mg conjugate per kg of animal. Mouse IgG-SAP (Cat. #IT-18) was used as a control. The results indicate that CD22 is a potential therapeutic target for cancer therapy.
Daniels-Wells TR, Helguera G, Rodriguez JA, Leoh LS, Erb MA, Diamante G, Casero D, Pellegrini M, Martinez-Maza O, Penichet ML (2013) Insights into the mechanism of cell death induced by saporin delivered into cancer cells by an antibody fusion protein targeting the transferrin receptor 1. Toxicol In Vitro 27(1):220-231. doi: 10.1016/j.tiv.2012.10.006
Summary: The antibody-avidin fusion protein ch128.1Av has been shown to target the human transferrin receptor 1 (TfR1) and kill malignant B cells by blocking the use of iron. Combination of this construct with a mono-biotinylated saporin custom conjugate produces an iron-independent toxicity to TfR1-expressing cells, even those that are resistant to ch128.1Av alone. The saporin-containing conjugate induces a transcriptional response consistent with oxidative stress and DNA damage. The data also show that the saporin conjugate is not toxic to human hematopoeietic stem cells.
Usage: An antibody-avidin fusion protein (ch128.1Av) was mixed with MonoBiotin-ZAP to make an immunotoxin that targets the human transferrin receptor 1 (TfR1).
Neuromedin B serves a role in nociceptive signaling.
Mishra SK, Holzman S, Hoon MA (2012) Neuromedin B serves a role in nociceptive signaling. Neuroscience 2012 Abstracts 471.22. Society for Neuroscience, New Orleans, LA.
Summary: We are interested in identifying new somatosensory signaling molecules and used an array based differential screen. In order to subtract genes not involved in signaling processes, we compared expression profiles in trigeminal ganglia (TG) with those of the geniculate ganglia (GG); a ganglia similar in structure but with different function. One gene we uncovered was neuromedin B (NMB), as expected from the differential expression, neuropeptide NMB is expressed in TG and dorsal root ganglia (DRG), but not in GG. Double labeling experiments, revealed NMB is expressed in a subset of sensory neurons that co[[unable to display character: ‐]]label with CGRP and TRPV1, suggestive of a role for NMB in nociception. Indeed, administration of NMB[[unable to display character: ‐]]antagonist greatly attenuates edema and nerve sensitization following stimulation of peripheral nerves with mustard oil, demonstrating that NMB contributes to neurogenic inflammation. Moreover, direct injection of NMB causes local swelling and nociceptive sensitization. Interestingly, we also found the receptor for NMB is expressed in interneurons in the superficial layers of the dorsal horn. We used NMB[[unable to display character: ‐]]saporin to specifically eliminate NMB-receptor expressing spinal cord cells and determined that they are required for responses to noxious heat, but not for reactions to mechanical and pruritic stimuli. Thus, NMB may be a neurotransmitter that is selectively involved in the perception of thermal stimuli, and has a role in neurogenic inflammation.
Kato J, Satake N, O’Donnell RT, Abuhay M, Lewis C, Tuscano JM (2013) Efficacy of a CD22-targeted antibody-saporin conjugate in a xenograft model of precursor-B cell acute lymphoblastic leukemia. Leuk Res 37(1):83-88. doi: 10.1016/j.leukres.2012.09.010
Summary: Most cases of acute lymphoblastic leukemia (ALL) are of B-cell lineage. Although children with ALL have a high survival rate, there is a subset of children with a much lower survival rate, and long-term side effects from treatment are problematic. CD22 has been suggested as a therapeutic target because it is not present on hematopoietic stem cells, therefore allowing regeneration of normal B cells following depletion of malignant B cells. The authors used a custom conjugate of the antibody HB22.7 and saporin to demonstrate specific toxicity against pre-B ALL cell lines. Mouse IgG-SAP (Cat. #IT-18) was used as a control.
Smith MA, Williams H, Krajewski SJ, Mcmullen NT, Rance NE (2011) Arcuate NK3 receptor-expressing KNDy neurons are essential for estrogen modulation of LH secretion and body weight in the female rat. Neuroscience 2011 Abstracts 712.07. Society for Neuroscience, Washington, DC.
Summary: Arcuate kisspeptin, neurokinin B, and dynorphin (KNDy) neurons have been proposed to mediate estrogen negative feedback in multiple species. To determine if these neurons are essential for this feedback, we ablated KNDy neurons in the arcuate nucleus of female rats using [MePhe7]Neurokinin B, a selective NK3 receptor (NK3R) agonist, conjugated to Saporin ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). The specificity of this conjugate for NK3R-expressing KNDy neurons is described in a separate abstract (see Krajewski et al., Soc. Neurosci. Abstr. 2011). Twenty-four female rats were ovariectomized (OVX) and received bilateral arcuate microinjections of either [MePhe7]NKB-SAP or a scrambled peptide conjugated to Saporin (Blank-SAP controls). 20-23 days later, animals were implanted with s.c. silastic capsules containing 17β-estradiol (E2), and animals were sacrificed 11 days later. Blood samples for RIA of serum LH were taken at time of OVX and injections (baseline), 20-23 days post-OVX, and 11 days after E2-treatment. Because OVX and E2-treatment have well-described effects on body weight, animals were weighed at the same three time points. In control animals, OVX induced a 13-fold rise in serum LH, which returned to baseline 11 days after E2 replacement. In contrast, OVX had no effect on serum LH in [MePhe7]NKB-SAP animals. There was a small decrease in serum LH 11 days after E2 replacement in [MePhe7]NKB-SAP animals, but the magnitude of this change was much less than seen in control animals. Control animals also exhibited a 20% increase in body weight 20-23 days after OVX, followed by a significant reduction after E2 replacement. Surprisingly, neither OVX nor E2 replacement affected body weight in [MePhe7]NKB-SAP-treated animals. Rather, these animals showed a steady increase in body weight throughout the experiment, at rates comparable to intact female rats or OVX rats treated with E2 (Williams et al., Endocrinology, 2010). Immunohistochemical studies showed near-complete destruction of KNDy neurons in the arcuate nucleus of [MePhe7]NKB-SAP animals. There was preservation of proopiomelanocortin and neuropeptide Y immunoreactivity in the arcuate nucleus and GnRH-immunoreactive fibers in the median eminence. These data provide compelling evidence that arcuate KNDy neurons play an essential role in estrogen negative feedback on LH secretion as well as the estrogen modulation of body weight.
Krajewski SJ, Smith MA, Williams H, Ciofi P, Lai JY, Mcmullen NT, Rance NE (2011) Ablation of NK3 receptor-expressing KNDy neurons in the rat arcuate nucleus using [MePhe7]Neurokinin B-Saporin. Neuroscience 2011 Abstracts 712.09. Society for Neuroscience, Washington, DC.
Summary: A subpopulation of neurons expressing kisspeptin, neurokinin B and dynorphin (KNDy neurons) has been shown to reside within the arcuate nucleus of many mammalian species. Although these peptides are critical for reproductive function, the precise role of the arcuate KNDy neurons is not fully understood. Here we describe a method to ablate KNDy neurons based on their co-expression of the Neurokinin 3 receptor (NK3R, Burke et al., J. Comp. Neurol, 2006). Saporin, a molecular neurotoxin, was conjugated to [MePhe7]Neurokinin B, a selective NK3R agonist ([MePhe7]NKB-SAP, Advanced Targeting Systems, San Diego, CA). Binding studies revealed that the conjugation of saporin did not alter the affinity of [MePhe7]NKB to NK3R in rat cerebral cortex membranes. To investigate the specificity of this conjugate for ablation of NK3R neurons, stereotaxic surgery was used to bilaterally inject [MePhe7]NKB-SAP into the arcuate nucleus of female rats. Control rats were injected with saporin conjugated to a scrambled peptide (Blank-SAP, Advanced Targeting Systems). Rats were sacrificed 31-34 days later and the brains were processed for immunohistochemical studies. Nissl stained sections from [MePhe7]NKB-SAP-treated rats showed no signs of inflammation at the injection sites and no qualitative changes in cell density compared to Blank-SAP control rats. Immunohistochemistry revealed near-complete loss of NK3R-immunoreactive (ir) neurons throughout the arcuate nucleus of [MePhe7]NKB-SAP rats. When the injection site was dorsal to the arcuate nucleus, there was also variable loss of NK3R-ir cells in the lateral hypothalamus and zona incerta. In the arcuate nucleus, [MePhe7]NKB-SAP injections resulted in a 98% and 94% reduction in the number of kisspeptin and neurokinin B-ir neurons, respectively, compared to Blank-SAP controls. The number of dynorphin-ir neurons in the arcuate nucleus of [MePhe7]NKB-SAP-treated rats was reduced by 67%, a value consistent with the co-expression of NK3R on dynorphin neurons in our previous study (Burke et al., J. Comp. Neurol, 2006). In contrast, arcuate proopiomelanocortin and neuropeptide Y immunoreactivity were preserved in [MePhe7]NKB-SAP rats. Moreover, there was no difference in GnRH-ir fiber density in the median eminence between the two groups. These results document the utility of [MePhe7]NKB-SAP for selective ablation of NK3R-expressing KNDy neurons in rat hypothalamus. These rats were used to examine the role of KNDy neurons in the estrogen regulation of LH secretion and body weight in the female rat (see Smith et al., Soc. Neurosci. Abstr. 2011).
Induction of CD4(+)CD25(+) T regulatory cells with CD103 depletion.
Zikri NN, Schumer E, Wang JJ, Gaughan A, Hadley GA, Moffatt-Bruce SD (2010) Induction of CD4(+)CD25(+) T regulatory cells with CD103 depletion. J Surg Res 163(1):162-168. doi: 10.1016/j.jss.2010.04.021
Summary: CD8+ T cells expressing CD103 have been shown to play a key role in the rejection of renal allografts. Use of M290-SAP (a custom saporin conjugation) allows allograft tolerance even in a completely mismatched islet cell transplant model. Use of 1 mg M290-SAP/kg body weight in mice allowed the authors to characterize the kinetics of M290-SAP and its induction of CD4 CD25 regulatory T cells.
An anti-CD103 immunotoxin promotes long-term survival of pancreatic islet allografts.
Zhang L, Moffatt-Bruce SD, Gaughan AA, Wang JJ, Rajab A, Hadley GA (2009) An anti-CD103 immunotoxin promotes long-term survival of pancreatic islet allografts. Am J Transplant 9:2012-2023. doi: 10.1111/j.1600-6143.2009.02735.x
Summary: The integrin CD103 is suspected of promoting organ allograft rejection and graft-versus-host disease. A custom conjugation was done between the non-depleting aCD103 antibody M290 and saporin. The conjugate was administered at 2.0 mg/kg to mice as an intraperitoneal injection (mouse IgG-SAP, Cat. #IT-18) was used as a control). The mice had previously received an islet transplant into a kidney capsule. Mice treated with M290-SAP were effectively depleted of CD103+ cells and had long term acceptance of the allografts.
Foehr ED, Lorente G, Kuo J, Ram R, Nikolich K, Urfer R (2006) Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model. Cancer Res 66(4):2271-2278. doi: 10.1158/0008-5472.CAN-05-1221
Summary: The receptor protein tyrosine phosphatase ß (RPTPß) is overexpressed in astrocytomas, and is a potential target for tumor therapy. After testing antibodies against an extracellular domain of RPTPß in vitro with Mab-ZAP (Cat. #IT-04), two custom conjugates, 7E4B11-SAP and 7A9B5-SAP, were created by Advanced Targeting Systems. The authors tested the custom conjugates, using anti-DAT-SAP (Cat. #IT-25) as a positive control, and mouse IgG-SAP (Cat. #IT-18) as a negative control. The 7E4B11-SAP conjugate displayed significant antitumor activity in mice engrafted with U87 glioma cells.
Gunhan-Agar E, Choudary P, Landerholm TE, Chalupa LM (2000) Depletion of cholinergic amacrine cells does not perturb the segregation of on and off cone bipolar cell projections. Neuroscience 2000 Abstracts 119.3. Society for Neuroscience, New Orleans, LA.
Summary: The pathways signaling onset and offset of light are segregated in the retina with On-cone and Off-cone bipolar cells terminating on the stratified dendrites of On and Off retinal ganglion cells (RGCs). During development the axons of On and Off cone bipolar cells form two strata in the IPL in a remarkably precise manner, without any obvious refinements. Moreover, such a precise ingrowth pattern occurs even after RGCs have been depleted (J. Neurosci., 2000, 201:306-314). Here we show by immunostaining that two bands of cholinergic processes are present in the rat retina as early as P1, some 7 days before the formation of segregated bipolar inputs. Double labeling of retinal sections with the antibody to recoverin (that recognizes On and Off cone bipolar cells) and the antibody for VACHT (which labels cholinergic processes) revealed that the segregated terminals of cone bipolar cells are juxtaposed with the two bands of cholinergic fibers. These observations suggested that the cholinergic fibers could serve as a scaffold for the later ingrowing bipolar cell axons. To test this hypothesis, we devised a novel method for depleting retinal cholinergic amacrine cells with a VACHT-saporin immunotoxin. A single treatment of the developing retina with this immunotoxin was found to eliminate virtually all cholinergic cells and processes. Recoverin labeling of bipolar cells showed that the axons of these neurons still form two stratified terminal bands within the IPL. Thus, neither RGCs nor cholinergic amacrine cell processes are required for the formation of segregated ON and Off cone bipolar cell projections.
Wu M, Shanabrough M, Leranth C, Alreja M (2000) Cholinergic excitation of septohippocampal GABA but not cholinergic neurons: implications for learning and memory. J Neurosci 20(10):3900-3908. doi: 10.1523/JNEUROSCI.20-10-03900.2000 PMID: 10804229
Summary: It has long been assumed that the drug-induced enhancement of learning and memory in both young and aged rats was accomplished through a cholinergic pathway in the hippocampus. Wu et al. used a fluorescent labeling molecule, 192-IgG conjugated to Cy3 (Custom Service from ATS) to visualize these neurons. They found that the effects of cognition-enhancing drugs are not facilitated through action on cholinergic neurons. Instead, activation of GABA neurons is implicated in this model.