Targeting Trends Newsletter 02q1

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Newsletter Highlights

  • Party Highlights (page 2)
  • Toxin Safety (page 5)
  • Serotonin Transporter Antibody (page 7)

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Control Conjugates: The Perfect Companion for Targeted Toxins – continued on page 6

Dr. Douglas Lappi, Chief Scientific Officer, Advanced Targeting Systems

Product information related to cover article: Control Conjugates Catalog

SFN 2001 Abstract Award Winner

ATS Customers Appreciated at Party

Recent Scientific References

Targeting Talk: Toxin Safety

  • We’re submitting a protocol to our IACUC to use IB4-SAP (Cat. #IT-10). We plan to inject the targeted toxin and then sacrifice the animal ten days later. What, if any, are the safety issues here?
  • Safety Instructions

Targeting Ticklers (Jokes)

Targeting Teaser Winners from last issue

Targeting Tools: Featured Products

  • Serotonin Transporter Antibody (Cat. #AB-N09)
  • L-Glutamine (Cat. #AB-T13)
  • NO-L-Glutamine (Cat. #AB-T14)

Targeting Technology Tutorial

Targeting Teaser (Jumble)

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Targeting Tools: Serotonin Transporter Antibody

Advanced Targeting Systems announces a new reagent for the study of the serotonergic systems—a monoclonal antibody to the serotonin transporter (Cat. #AB-N09). This murine monoclonal is made with a peptide from an extracellular domain of rat serotonin re-uptake transporter (SERT), and thus is able to attach to cells that express SERT. Homology with the human form is very high, but very low for other transporters such as the norepinephrine transporter (NET) and dopamine transporter (DAT). Figure 1 shows FACS analysis of human platelets, which express the transporter. There is a strong shift with antibody labeled with FITC- second antibody conjugate. FACS analysis also shows no interaction with cells expressing NET or DAT, as is expected from the homology cited above. Figure 2 demonstrates excellent immunostaining by the antibody of the rat raphe nucleus, a major site of SERT expression.
This antibody is a powerful new tool for systems that are important in several biological processes, as the market sales of the anti-depressant drug fluoxetine demonstrate. It is expected, and our preliminary data are confirming, that this antibody will also be excellent for in vivo targeting of SERT-positive neurons.

Targeting Talk: Toxin Safety

Safety Instructions

Good laboratory technique must be employed for the safe handling of this product. This requires observation of the following practices:

  1. Wear appropriate laboratory attire, including lab coat, gloves and safety glasses.
  2. Do not pipet by mouth, inhale, ingest or allow product to come into contact with open wounds. Wash thoroughly any part of the body which comes into contact with the product.
  3. Avoid accidental autoinjection by exercising extreme care when handling in conjunction with any injection device.
  4. This product is intended for research use by qualified personnel only. It is not intended for use in humans or as a diagnostic agent. Advanced Targeting Systems is not liable for any damages resulting from the misuse or handling of this product.

For disposal: autoclave, or expose to 0.2 M NaOH, materials that come into contact with the toxin.

We’re submitting a protocol to our IACUC to use IB4-SAP (Cat. #IT-10). We plan to inject the targeted toxin and then sacrifice the animal ten days later. What, if any, are the safety issues here?

The only danger to lab personnel from IB4-SAP would be accidental self-injection, and even then, at the doses typically used in rats, it would only produce very localized effects at the injection site.

Once injected into animals, the agent is rapidly rendered inaccessible to anyone else by binding, internalization and eventual catabolism. It is extremely unlikely that intact toxin would ever be excreted or recoverable from the rats. The components of the toxin, IB4 and saporin, by themselves are no toxic threat. We use no special precautions with such rats except appropriate care for whatever neurologic deficits they develop, i.e. foot drop, autotomy, etc.

One caveat: To the best of my knowledge the above statements are accurate, but I do not know of any experimental data that directly addresses the issues. I base my comments on our long experience with similar agents including ricin and volkensin which are much more toxic and unstable.

Your recent issue of Targeting Trends (see Jul-Aug-Sep, 2001) stated that it was unlikely that saporin compounds or constituents would be excreted in urine or feces. However, you acknowledge that experimental data is lacking. Have there been any tests of animal urine or feces for saporin content? My animal care staff are concerned.

One of the reasons that no studies have been done on excretion of saporin is that there isn’t much on the theoretical side to cause concern. The primary issue is that the quantity used in mice (and even rabbits) is so small that when looked at in human terms (i.e., an animal 10 to 100-times larger), the dosage becomes insignificant. The LD50 for saporin in mice is 4-8 mg/kg; that would translate in humans to more than you’ll ever use! The immunotoxins, which contain only about 20% saporin by weight, really do not contain all that much saporin.

Looking at it another way, you need a concentration of about 100 nM to see even a vague hint of toxicity of saporin to cells. In human blood, that would correspond to 24 mg injected systemically into a person. It would be really expensive for anyone to get close to that number.

As far as urine and feces goes, the same calculations are appropriate, but there will be considerable degradation – the protein content in urine and feces is quite low and the probability is that you will be dealing with only saporin. Remember saporin is a plant protein that is related to proteins in foods that we eat (cucumbers, for example).

Reference
Stirpe F, Derenzini M, Barbieri L, Farabegoli F, Brown AN, Knowles PP, Thorpe PE (1987) Hepatotoxicity of immunotoxins made with saporin, a ribosome-inactivating protein from Saponaria officinalis. Virchows Arch [B] 53:259-271.

 
Are there any studies which indicate what doses of saporin (by itself or compounded with an antibody) would be hazardous if ingested or injected (i.e. systemic dose level resulting in death or organ dysfunction).

When there is an antibody that does recognize a human epitope (the human p75-saporin immunotoxin that is used in rabbits, for example), at about 1 pM one sees the slightest bit of toxicity to cells. That translates, if injected by error into a human blood supply, to about 170 micrograms. That also is a gigantic dose. I am using very conservative numbers here, and the bottom line is that you cannot accidentally reach such dangerous levels under normal handling situations.

Having said all this, we still recommend that our customers take excellent care of themselves and we state clearly that precautions should be taken by people handling these materials, just as they should use precautions with all laboratory chemicals. Please refer to the data sheets provided with our products for safety instructions.

See also: Targeted Toxins Catalog

Targeting Topics 02q1

Colocalization of mu-opioid receptors and activated G-proteins in rat cingulate cortex.

Vogt LJ, Sim-Selley LJ, Childers SR, Wiley RG, Vogt BA.

J Pharmacol Exp Ther 299(3):840-848, 2001. PMID: 11714867

The anterior cingulate cortex (ACC) is a primary site of opiate drug action, and much of this activity is associated with the μ-opioid receptor (MOR). The mechanisms by which MOR regulates pain in the ACC are not well understood. Using anti- DBH-SAP (7 μg into left lateral ventricle in rat; Cat. #IT-03) the authors mapped MOR activity in the ACC and evaluated the histochemical and behavioral relationships between MOR binding and μ-receptor- activated G-proteins after lesioning.

Selective immunolesions of cholinergic neurons in mice: effects on neuroanatomy, neurochemistry, and behavior.

Berger-Sweeney J, Stearns NA, Murg SL, Floerke-Nashner LR, Lappi DA, Baxter MG.

J Neurosci 21(20):8164-8173, 2001. PMID: 11588189

192-Saporin (Cat. #IT-01) has long been an effective agent for elimination of cholinergic neurons in the basal forebrain of rats. Until the development of mu p75-SAP (Cat. #IT-16) there was no equivalent agent for use in mice. The authors tested mu p75-SAP in vitro and in vivo (1.8-3.6 μg in right lateral ventricle), using cytotoxic, histochemical, and behavioral assays. The data shows that mu p75-SAP is a highly selective and efficacious lesioning agent for cholinergic neurons in the mouse. The authors conclude that mu p75- SAP will be a powerful tool to use in combination with genetic modification to investigate cholinergic damage in mouse models of Alzheimer’s disease.

Extensive immunolesions of basal forebrain cholinergic system impair offspring recognition in sheep.

Ferreira G, Meurisse M, Gervais R, Ravel N, Levy F.

Neuroscience 106(1):103-116, 2001. PMID: 11564421

Through the use of 192-Saporin (Cat. #IT-01) the association of basal forebrain cholinergic neurons to learning instrumental tasks has been well established in the rat. The authors wished to examine whether these neurons were also associated with social learning tasks, such as offspring recognition in sheep. Using ME20.4-SAP (Cat. #IT-15) the basal forebrain cholinergic neurons of sheep were lesioned by intraventricular bilateral injections (150 μg). The results demonstrate that these neurons contribute to visual discrimination learning, and are involved in formation of lamb recognition memory.

Dissociation between the attentional functions mediated via basal forebrain cholinergic and GABAergic neurons.

Burk JA, Sarter M.

Neuroscience 105(4):899-909, 2001. PMID: 11530228

The specificity and efficacy of 192-Saporin (Cat. #IT-01) has allowed the extensive investigation of cortical cholinergic inputs in attentional functions. Little is known about the function of non-cholinergic neurons because of the lack of a specific tool to eliminate these projections. The authors injected 192-Saporin (0.1 μg/0.5 μl bilateral infusions) into rats and compared performance to rats treated with ibotenic acid to eliminate GABAergic neurons in attention performance tasks. While the ibotenic acid lesions were not as specific as those produced by 192-Saporin, the data suggest a role for the basal forebrain GABAergic neurons in attentional functions.

Novel method for localized, functional sympathetic nervous system denervation of peripheral tissue using guanethidine.

Demas GE, Bartness TJ.

J Neurosci Methods 112(1):21-28, 2001. PMID: 11640954

Sympathectomy, or surgical interruption of sympathetic nerve pathways, is an important technique in the analysis of the sympathetic nervous system. The authors investigate and compare several different methods of performing a sympathectomy in hamsters, including surgery, chemical, and immunotoxic lesions using anti- DBH-SAP (ten 2-μl injections, at either 0.65 μg/μl or 0.325 μg/μl, into inguinal white adipose tissue; Cat. #IT-03).

Macrophage-derived IL-18-mediated intestinal inflammation in the murine model of Crohn’s disease.

Kanai T, Watanabe M, Okazawa A, Sato T, Yamazaki M, Okamoto S, Ishii H, Totsuka T, Iiyama R, Okamoto R, Ikeda M, Kurimoto M, Takeda K, Akira S, Hibi T.

Gastroenterology 121(4):875-888, 2001. PMID: 11606501

Crohn’s disease is an inflammatory bowel disease that is associated with several changes in the immune system, including an increased number of infiltrating macrophages. These macrophages release a variety of cytokines that are responsible for inflammation. The authors investigated the role of these macrophages in a mouse model by eliminating them with Mac-1-SAP (20 μg parenterally in tail vein; Cat. #IT-06). Seven days after treatment, mice showed no evidence of intestinal inflammation. These data demonstrate the role of macrophages in the development of inflammatory bowel conditions.

The effects of manipulations of attentional demand on cortical acetylcholine release.

Himmelheber AM, Sarter M, Bruno JP.

Brain Res Cogn Brain Res 12(3):353-370, 2001. PMID: 11689296

Cortical cholinergic afferents from the basal forebrain are suspected to be involved in attentional tasks. Regulatory impairment of these afferents has been hypothesized to contribute to attentional deficits seen in conditions as diverse as Alzheimer’s disease and schizophrenia. The authors have previously shown that 192-Saporin (Cat. #IT-01) lesions result in severe impairments in tasks requiring sustained attentional processing. In these experiments the authors suggest that cell response is dependent on the level of demand. They demonstrate that removal of p75+ cells (0.5 μg/μl bilaterally infused into the nucleus basalis region in rat) impairs sustained attentional performance, but does not impact low-demand task performance.

Long-term intrathecal catheterization in the rat.

Jasmin L, Ohara PT.

J Neurosci Methods 110(1-2):81-89, 2001. PMID: 11564527

The authors have developed a method that allows repeated administration of drugs with minimal stress to an experimental animal. To test the efficacy of this intrathecal catheter, they injected anti-DBH-SAP (5 μg; Cat. #IT-03) and investigated the noradrenergic denervation of the spinal cord. All animals treated with anti-DBH-SAP showed extensive loss of spinal noradrenergic ennervation. Even three months after catheter implantation, the elimination of noradrenergic neurons in the spinal cord could be produced. This indicates the intrathecal catheter is an effective tool for the study of multiple-dose drug delivery.

Differential changes in rat cholinergic parameters subsequent to immunotoxic lesion of the basal forebrain nuclei.

Waite JJ, Chen AD.

Brain Res 918(1-2):113-120, 2001. PMID: 11684049

192-Saporin (Cat. #IT-01) is used extensively to eliminate the cholinergic neurons of the basal forebrain in rats. Waite and Chen compare the degree of loss between 192-Saporin (6 or 8.2 μg in 10 μl into left lateral ventricle) and control (Saporin, 1.82 μg into left lateral ventricle; Cat. #PR-01) using three methods: Assay of post mortem choline acetyltransferase activity, in vivo microdialysis of extracellular acetylcholine (ACh), and in vivo assessment of the rate of ACh synthesis. The infusion of saporin alone had no effect. After fifteen weeks, the authors report compensation of cholinergic activity in lesioned animals occurs in the hippocampus, but not in the frontal cortex as determined by measurement of the rate of ACh synthesis.

Cover Article: Control Conjugates – The Perfect Companion for Targeted Toxins

Dr. Douglas Lappi, Chief Scientific Officer, Advanced Targeting Systems

The field of targeted toxins has made enormous strides in the years since Advanced Targeting Systems introduced the first research targeted toxin in 1994. We now offer 16 different targeted toxins, with more on the way. The number of important papers in high impact journals continues to rise, and the sophistication of the studies is very impressive.

Much of the research using targeted toxins has been dedicated to characterizing the lesions caused by these molecules. We studied this important work to understand where ATS could be most helpful. The result of our analysis is the offering this year of new control molecules that chemically resemble the targeted toxins, but are not targeted to any cell type.

Saporin alone (not conjugated) has been used by some researchers as a control. But without a stand-in for the targeting agent, there are fundamental differences in physiochemical structure. The new controls solve this problem. Some researchers have included the targeting agent mixing the antibody or peptide with saporin. While this has the components of the targeted toxin, it does not include the structural features of the conjugation chemistry. The new controls solve this difficulty.

ATS targeted toxins fall into three categories: immunotoxins, ligand toxins and second immunotoxins. To provide researchers with more verifiable targeting tools, each of these now has a dedicated control.

Immunotoxins: This category can be further divided into those with antibodies that are based on mouse monoclonals (192-Saporin, OX7- SAP,anti-DBH-SAP,ME20.4-SAP), and those with antibodies based on rat monoclonals (Mac-1-SAP, mu p75-SAP).

For the mouse monoclonals, we offer Mouse-IgG-SAP (Cat. #IT-18), produced by conjugation of mouse IgG to saporin. Mouse IgG replaces the murine monoclonal IgG that performs the specific targeting, and saporin is used in both. The molecular structure of the two is similar, even to the chemistry of conjugation. The only difference is the replacement of the targeting agent with mouse IgG that has no target. Figure 1 shows the same lack of targeted cytotoxicity as saporin alone and approximately 1000-fold less than targeted material.

The rat immunotoxins also have a negative control. Rat IgG-SAP, Cat. #IT-17, is made with rat IgG coupled to saporin with the same chemistry. Figure 2 shows that rat IgG-SAP has no more cytotoxicity to cells than saporin alone, while the targeted toxin is more than four orders of magnitude more toxic.

For the second immunotoxins (Mab-ZAP, Rab-ZAP), use Goat IgG- SAP, Cat. #IT-19. It works on the same easy principle as Mouse IgG- SAP and Rat IgG-SAP.

Ligand-toxins: Blank-SAP (Cat. #IT-21) is constructed from a nonsense peptide, a re-arranged alpha melanocyte- stimulating hormone. This sequence contains amino acids common to the ligands of G protein-coupled receptors, but has no known homology. Like all of our peptide- toxins, it has a 1:1 molar ratio of saporin to peptide, and is void of any free peptide or non-conjugated saporin. It’s a perfect match to the peptide ligand-toxins SP-SAP, SSP- SAP, orexin-SAP, dermorphin-SAP, and CRF-SAP. Figure 3 is an illustration of Blank-SAP “shooting blanks” relative to a targeted toxin. The targeted toxin is more than two orders of magnitude more potent than Blank-SAP or saporin alone.

The great thing about these new controls is they are ready to use. You don’t have to make strange calculations to figure how much of each component to add; you just use the same amount of control as you do the targeted toxin, and that’s it! Couldn’t be much simpler.