Targeting Trends Newsletter 02q2

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Newsletter Highlights

  • It’s Twins!(page 2)
  • Toxin Safety (page 5)
  • OX7-SAP (page 7)

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HCRT-SAP Lesion Produces Sleepiness While anti-DBH-SAP Lesion Does Not  – continued on page 2 and page 6

Carlos Blanco-Centurion contributes this report resulting from work done for an abstract at the 2001 Society for Neuroscience meeting: Hypocretin B-Saporin Lesions of the Brainstem Increase REM Sleep at Night CA Blanco- Centurion, RJ Salin-Pascual, D Gerashchenko, MA Greco, PJ Shiromani. Harvard Medical School & UNAM, Mexico City. This abstract won the ATS Abstract Award. Note: HCRT-SAP is also known as orexin-SAP.

Product information related to cover article: Anti-DBH-SAP (Cat. #IT-03)

SP-SAP (Cat. #IT-07) Licensed to Cell Targeting Technologies

Matthew Kohls Welcomes the Diapered Duo

Recent Scientific References

Targeting Talk: Toxin Safety

  • Your recent issue of Targeting Trends 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.
  • 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).

Targeting Ticklers (Jokes)

Targeting Teaser Winners from last issue

Targeting Tools: OX7 and OX7-SAP

Targeting Technology Tutorial

Targeting Teaser (Jumble)


Targeting Tools: OX7 and OX7-SAP

OX7-SAP is a cytotoxin to many types of neurons. It is one of the most potent of the saporin immunotoxins— the conjugates between a cell surface antibody and the ribosome-inactivating protein from Saponaria officinalis. The reason for the potency could be due to the construct’s grand tradition. The antibody, OX7, is one of the original antibodies to cell surface proteins produced by England’s Medical Research Council, the famous MRC of Crick, Perutz, Brenner and many others. This antibody was made by injection of rat white blood cells for immunization and recognizes CD90 or Thy 1. Thy 1 is expressed on virtually all neurons after cessation of axonal growth.1 The mouse monoclonal recognizes rat and mouse Thy 1. Figure 1 illustrates analysis by flow cytometry of PC12 cells with OX7 and demonstrates a remarkable shift.

[threecol_two][/threecol_two] [threecol_one_last]Figure 1. Analysis by flow cytometry of PC12 cells with OX7[/threecol_one_last]

To the neuroscience community, OX7-SAP has a distinguished career. It was used by Ron Wiley and his colleagues in the first applications of immunotoxins in the nervous systems. He used it as a “suicide transport” agent, in which the injection is at a terminal site with the purpose of eliminating the cell that projects to that point through uptake at the terminal, transport to the cell body and eventual death by protein synthesis inhibition.2 He also used OX7-SAP by intraventricular injection for the subsequent removal of Purkinje cells.3 OX7-SAP has been used by numerous investigators in variations on this theme. The immunotoxin has also been used to deplete mouse blood of T lymphocytes, because these also express Thy 1.4 (See the ATS website for a complete list of references.)

[threecol_two][/threecol_two] [threecol_one_last]Figure 2. Cytotoxicity of two different lots of OX7-SAPand of non-conjugated saporin (SAP) to PC12 cells in culture. Cells were plated at 5000 cells per well and allowed to acclimate. Samples were added at the indicated concentrations and cells were incubated for 72 hours. MTS (Promega) was added and, after color development, wells were read with a Molecular Dynamics SpectraMax 340. ED50’s of each compound are color-coded. Data analysis is by PRISM by GraphPad. [/threecol_one_last]

Figure 2 shows the cytotoxicity of OX7-SAP to PC12 cells in culture. There is a greater than 4 orders of magnitude difference between the non-conjugated saporin and the OX7-SAP which is targeted to PC12 cells because of their Thy 1 expression.

For an excellent discussion on the many applications for OX7-SAP in lesioning of neurons, see the recent review by Wiley and Kline.5


  1. Tiveron MC, Barboni E, Pliego Rivero FB, Gormley AM, Seeley PJ, Grosveld F, Morris R, Berger-Sweeney J, Stearns NA, Murg SL, et al. (1992) Selective inhibition of neurite outgrowth on mature astrocytes by Thy-1 glycoprotein. Nature 355:745-748.
  2. Wiley RG, Stirpe F, Thorpe P, Oeltmann TN (1989) Neuronotoxic effects of monoclonal anti-Thy 1 antibody (OX7) coupled to the ribosome inactivating protein, saporin, as studied by suicide transport experiments in the rat. Brain Res 505:44-54.
  3. Davis TL, Wiley RG (1989) Anti-Thy-1 immunotoxin, OX7-saporin, destroys cerebellar Purkinje cells after intraventricular injection in rats. Brain Res 504:216-222.
  4. Marcucci F, Lappi DA, Ghislieri M, Martineau D, Siena S, Bregni M, Soria M, Gianni AM (1989) In vivo effects in mice of an anti-T cell immunotoxin. J Immunol 142:2955-2960.
  5. Wiley RG, Kline IR (2000) Neuronal lesioning with axonally transported toxins. J Neurosci Meth 103:73-82.

Targeting Talk: Toxin Safety

Q: Your recent issue of Targeting Trends 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.

A: 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;1 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).

Reference1. 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.


Q: 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).

A: 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.

Targeting Topics 02q2

GABAergic septohippocampal neurons are not necessary for spatial memory.

Pang KC, Nocera R, Secor AJ, Yoder RM.

Hippocampus 11(6):814-827, 2001. PMID: 11811676

The medial septum and diagonal band of Broca (MSDB) are necessary for spatial memory. Both cholinergic and GABAergic neuronal populations are present in the MSDB. 192-Saporin (Cat. #IT-01) was used to eliminate cholinergic populations and kainic acid was used to reduce numbers of GABAergic neurons. Both agents were injected (independently or in combination) into the medial septum and each diagonal band of rats (192-Saporin 250 ng MS, 150 ng DB) to determine the importance of GABAergic neurons in the MSDB for spatial memory. The results showed elimination of GABAergic neurons has no impact on spatial memory, while elimination of cholinergic neurons has a mild impact.

Selective immunolesioning of cholinergic neurons in nucleus basalis magnocellularis impairs prepulse inhibition of acoustic startle.

Ballmaier M, Casamenti F, Zoli M, Pepeu G, Spano P.

Neuroscience 108(2):299-305, 2001. PMID: 11734362

One of the measures for schizophrenia is a deficit in sensorimotor gating (the ability of the brain to filter sensory input to focus on selective stimuli) measured by prepulse inhibition (PPI) of the startle reflex. The authors injected 300 nl of 400 ng/μl 192-Saporin (Cat. #IT-01) into each side of the nucleus basalis magnocellularis (NBM) in rats to examine the effect of NBM cholinergic neuron elimination on the startle reflex. The data show a significant, persistent disruption of the PPI independent of the amplitude of the startle reflex. This suggests the NBM may play an important role in information processing in schizophrenia.

Lack of effect of moderate Purkinje cell loss on working memory.

Wrenn CC, Wiley RG.

Neuroscience 107(3):433-445, 2001. PMID: 11718998

When 192-Saporin (Cat. #IT-01) is injected intracerebroventricularly, some p75-expressing cerebellar Purkinje cells are eliminated along with cholinergic neurons. To verify that the effects of basal forebrain lesions on working memory were not caused by loss of these Purkinje cells the authors compared doses of 1 μg OX7-SAP (Cat. #IT-02) and either 2 μg or 4 μg of 192-Saporin injected into the lateral ventricle. The data show that although similar amounts of Purkinje cells were eliminated by OX7-SAP and the lower dose of 192- Saporin, no working memory deficits resulted. Only the 4-μg dose of 192- Saporin produced working memory deficits, they conclude that this is not due to Purkinje cell loss, but the loss of cholinergic neurons.

Central cholinergic depletion induced by 192 IgG-saporin alleviates the sedative effects of propofol in rats.

Pain L, Jeltsch H, Lehmann O, Lazarus C, Laalou FZ, Cassel JC.

Br J Anaesth 85(6):869-873, 2000. PMID: 11732522

In order to examine the effect of cholinergic depletion on the sedative potency of propofol in rats the authors injected 1 μg of 192-Saporin (Cat. #IT-01) into each lateral ventricle. The findings indicate a ~50% reduction in sedative potency in lesioned rats.

Transverse patterning reveals a dissociation of simple and configural association learning abilities in rats with 192 IgG-saporin lesions of the nucleus basalis magnocellularis.

Butt AE, Bowman TD.

Neurobiol Learn Mem 77(2):211-233, 2002. PMID: 11848720

Using 80 ng bilateral infusions of 192-Saporin (Cat. #IT-01) into each of the medial and lateral target sites of the nucleus basalis magnocellularis (NBM) in rats, the authors demonstrate that lesioning the cholinergic systems of the NBM impairs a more complicated learning technique, while leaving simple association learning intact. The results also show that the transition between these two learning strategies is impaired in lesioned animals.

Facilitation of dopamine-mediated locomotor activity in adult rats following cholinergic denervation.

Mattsson A, Ogren SO, Olson L.

Exp Neurol 174(1):96-108, 2002. PMID: 11869038

It has been hypothesized that hyperactivity of dopaminergic systems is involved in schizophrenia. The authors examine the effect of central cholinergic denervation on dopamine-mediated functions. Adult rats received bilateral injections of 2.5 μg of 192-Saporin (Cat. #IT-01) into the lateral ventricles. Neonatal rats received 400 ng 192-Saporin in bilateral i.c.v. injections. The results indicate that severe cholinergic deficiencies in the forebrain can lead to dopaminergic overfunction, one of the possible mechanisms in schizophrenia.

Isoflurane and nociception: spinal alpha2A adrenoceptors mediate antinociception while supraspinal alpha1 adrenoceptors mediate pronociception.

Kingery WS, Agashe GS, Guo TZ, Sawamura S, Davies MF, Clark JD, Kobilka BK, Maze M.

Anesthesiology 96(2):367-374, 2002. PMID: 11818770

The authors injected 3 μg/3 μl of anti-DBH-SAP (Cat. #IT-03) into the lateral ventricle of rats to determine whether noradrenergic brainstem nuclei and descending spinal pathways are responsible for the antinociceptive actions of isoflurane. The results indicate that isoflurane modulates nociception by as many as three mechanisms, utilizing various combinations of noradrenergic neurons, adrenoceptors, and descending spinal pathways.

Cytotoxic targeting of isolectin IB4-binding sensory neurons.

Vulchanova L, Olson TH, Stone LS, Riedl MS, Elde R, Honda CN.

Neuroscience 108(1):143-155, 2001. PMID: 11738138

Vulchanova et al. examine the role of IB4-binding neurons in nociception. IB4-SAP (Cat. #IT-10) was injected into rats (2 μg in left sciatic nerve). The resulting ablation of IB4-binding neurons provides evidence for their role in nociceptive processing and demonstrates a rapid compensatory response to signalling of acute pain.

Lesions of the nucleus basalis magnocellularis induced by 192 IgG-saporin block memory enhancement with posttraining norepinephrine in the basolateral amygdala.

Power AE, Thal LJ, McGaugh JL.

Proc Natl Acad Sci U S A 99(4):2315-2319, 2002. PMID: 11830635

There is evidence that memory consolidation (retention) can be modulated by drugs and stress hormones acting in the basolateral amygdala (BLA). The BLA sends projections to the nucleus basalis magnocellularis (NBM), which in turn sends cholinergic projections to the neocortex. The authors used 100 ng bilateral infusions of 192-Saporin (Cat. #IT-01) in 500 nl 0.1 M PBS to investigate whether lesions of the cholinergic NBM projections affect BLA modulation of memory. 192- Saporin lesions blocked memory enhancement normally induced by norepinephrine infusions into the BLA. This finding suggests NBM- cortex projections may mediate BLA modulation of memory storage or processing in the neocortex.

Cholinergic and noncholinergic septal neurons modulate strategy selection in spatial learning.

Cahill JF, Baxter MG.

Eur J Neurosci 14(11):1856-1864, 2001. PMID: 11860481

The authors compared ibotenic acid (IA)-treated rats with those injected with 45 ng and 30 ng of 192-Saporin (Cat. #IT-01) into two separate coordinates of the medial septum/vertical limb of the diagonal band (MS/VDB) to investigate the role of basal forebrain projections in modulating strategy selection in spatial learning. While rats with IA lesions in the MS/VDB demonstrated significant disruption of the learning process, the 192-Saporin-lesioned rats did not show this effect.

SP-SAP licensed to Cell Targeting Technologies

Advanced Targeting Systems has exclusively licensed the Substance P- Saporin (SP-SAP) compound to Cell Targeting Technologies for development as a pharmaceutical agent. ATS and collaborators at University of Minnesota and University of California, San Diego have completed several preclinical studies using SP-SAP and have thus far found it safe and effective in two animal models. The journal Science reported the results of studies in a rat chronic pain model showing that SP-SAP appears to eliminate chronic pain permanently while not interfering with the perception of normal, acute pain sensations. Cell Targeting Technologies will raise capital to fund in-depth toxicology studies and plans to bring SP- SAP to clinical trial within the next two years.

Cover Article: HCRT-SAP Lesion Produces Sleepiness While anti-DBH-SAP Lesion Does Not

Carlos Blanco-Centurion contributes this report resulting from work done for an abstract at the 2001 Society for Neuroscience meeting: Hypocretin B-Saporin Lesions of the Brainstem Increase REM Sleep at Night CA Blanco- Centurion, RJ Salin-Pascual, D Gerashchenko, MA Greco, PJ Shiromani. Harvard Medical School & UNAM, Mexico City. This abstract won the ATS Abstract Award. Note: HCRT-SAP is also known as orexin-SAP (Cat. #IT-20).

Loss of hypocretin (HCRT, orexin) neurons has been linked to narcolepsy.1 These neurons project widely throughout the brain,2 but it is not known which projection to which target site produces what symptom of narcolepsy. Recently we showed that HCRT receptors are present in brainstem areas implicated in REM sleep.3 Since abnormal triggering of REM sleep characterizes narcolepsy, we have used HCRT-SAP, a targeted toxin that selectively lesions HCRT-2 receptor- bearing cells, to assess the effect on sleep. We also used anti-DBH-SAP to destroy specifically noradrenergic neurons of the locus coeruleus (NA-LC), which are the major brainstem output of HCRT neurons. We knew that hypocretin neurons project to the pons but the goal of this study was to determine which pontine neurons regulate sleep.

[threecol_one][/threecol_one] [threecol_one][/threecol_one] [threecol_one_last][/threecol_one_last]

[threecol_one][/threecol_one] [threecol_two_last]Figure 1.
Panel A=saline, B=unilateral lesion with HCRT-SAP, C=bilateral lesions with HCRT-SAP.
D and E are magnifications showing the effect of saline and anti-DBH-SAPmicroinjections respectively. Arrows indicate NADPH+ cells (cholinergic)[/threecol_two_last]

Methodology. Rats were implanted with electrodes for polysomnographic recordings. At the same time rats received either a bilateral single microinjection of pyrogen-free saline solution or anti-DBH-SAP (1 mg/mL; vol=80 nL) aimed to the LC nuclei. Afterward, polysomnographic recordings were done across 24 h on the 3rd, 6th, 9th, 12th and 18th days post-injection (12:12h lights on/off). Scoring was made visually on a computer (Icelus software) in 12s epochs for waking, slow wave sleep (SWS) and REM sleep by one technician blind to treatment. ANOVA and t-test with Bonferroni correction (where appropriate) were used to compare changes in sleep parameters. After recordings were done, subjects were sacrificed, brains fixed, removed and sectioned for immunohistochemistry staining for DBH (1:50K). Histochemistry for NADPH was made as well. A blind- treatment technician counted DBH-ir and NADPH+ cells across mesopontine tegmentum in a 1:5 sections protocol.

The experimental protocol was similar to anti-DBH-SAP lesions for HCRT-SAP (100 ng/mL), except it was microinjected lateral to LC and in larger volume (500 nL). In addition to DBH and NADPH labeling, brain sections were immunostained for the specific nuclear protein NeuN to outline the cell loss area. In all cases avidin- biotin-DAB method was used to visualize the antigens.

[twocol_one]Figure 2[/twocol_one] [twocol_one_last]Figure 3[/twocol_one_last]

Results. Post mortem analysis revealed anti-DBH-SAP destroyed noradrenergic neurons in the LC but spared NADPH+ cells (Figs. 1,2). The anti-DBH-SAP pontine lesions did not affect sleep either during day or night period (Fig. 3). This suggests noradrenergic LC neurons are not important in maintaining wakefulness. In contrast, HCRT-SAP injections into the dorsolateral pons increased significantly both SWS and REM sleep. This effect was only present during the normal rat active period (night), but not so during the rest period. The sleepiness induced by HCRT-SAP pontine lesions is mainly generated by an increase of the sleep drive, that is, an increase in the number of episodes at night (p<0.05). Sleepiness was also associated with a significant reduction in the number of NeuN-ir within the parabrachial complex as well as NADPH+ cells in the LDTg (laterodorsal tegmental). Yet HCRT- SAP did not affect DBH-ir (Figs. 4, 5). Our results suggest both parabrachial and LDTg cholinergic neurons, but not noradrenergic LC neurons, express the hypocretin-2 (orexin B) receptor; anti-DBH-SAP only affects noradrenergic LC neurons. Additionally these HCRT- SAP sensitive neurons in the dorsolateral pons seem to be involved in sustaining waking during the normal active period. As a result, the daytime sleepiness observed in narcoleptic HCRT-deficient subjects could be associated with a lack of excitatory input to parabrachial and LDTg rather than to LC neurons, as has been proposed.

[fourcol_one][/fourcol_one] [fourcol_one][/fourcol_one] [twocol_one_last]Figure 4. Panel A=saline, Panel B=HCRT-saporin (100 ng/μl, TV=500 nl). Both sections (50 μm=thickness) were immunostained to label the specific neuron’s nuclear protein NeuN. Magnification in both cases is 4X. 4V=Fourth ventricle, LC=Locus coeruleus, PB=Parabrachial nuclei.[/twocol_one_last]
[sixcol_five]Figure 5. HCRT-SAP(100 ng/μl; vol=500 nl) spares DBH-IR cells in the LC (A) but destroys NADPH+ cells (cholinergic) in the LDTg (C). (B=saline; C=HCRT-SAP). [/sixcol_five] [sixcol_one_last].[/sixcol_one_last]

[twocol_one][/twocol_one] [fourcol_one][/fourcol_one] [fourcol_one_last][/fourcol_one_last]


  1. Nishino S, Ripley B, Overeem S, Lammers GJ, Mignot E (2000) Lancet 355:39-40.
  2. Peyron C, Tighe DK, Van de Pol AN, de Lecea L, Heller HC, Sutcliffe JG, Kilduff TS (1998) J Neurosci 18:9996-10015.
  3. Greco MA, Shiromani PJ (2001) Mol Brain Res 88:176-182.
  4. Gerashchenko D, Kohls MD, Greco M, Waleh NS, Salin-Pascual R, Kilduff TS, Lappi DA, Shiromani PJ (2001) J Neurosci 21:7273-7283.

Related Production Information: Orexin-SAP (Cat. #IT-20), Anti-DBH-SAP (Cat. #IT-03)