Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor.

Substance P is released in the spinal cord in response to painful stimuli, but its role in nociceptive signaling remains unclear. When a conjugate of substance P and the ribosome-inactivating protein saporin was infused into the spinal cord, it was internalized and cytotoxic to lamina I spinal cord neurons that express the substance P receptor. This treatment left responses to mild noxious stimuli unchanged, but markedly attenuated responses to highly noxious stimuli and mechanical and thermal hyperalgesia. Thus, lamina I spinal cord neurons that express the substance P receptor play a pivotal role in the transmission of highly noxious stimuli and the maintenance of hyperalgesia.

Transmission of chronic nociception by spinal neurons expressing the substance P receptor.

Substance P receptor (SPR)-expressing spinal neurons were ablated with the selective cytotoxin substance P-saporin. Loss of these neurons resulted in a reduction of thermal hyperalgesia and mechanical allodynia associated with persistent neuropathic and inflammatory pain states. This loss appeared to be permanent. Responses to mildly painful stimuli and morphine analgesia were unaffected by this treatment. These results identify a target for treating persistent pain and suggest that the small population of SPR-expressing neurons in the dorsal horn of the spinal cord plays a pivotal role in the generation and maintenance of chronic neuropathic and inflammatory pain.

Activity of a monoclonal antibody-saporin-6 conjugate against B-lymphoma cells.

A monoclonal antibody reactive with the immunoglobulin heavy chain (TEC IgM) has been conjugated to saporin-6 (SAP), which is the major ribosome-inactivating protein from the seeds of the plant Saponaria officinalis. Studies with Burkitt's lymphoma cell line Bjab 113 demonstrate that this immunotoxin is capable of killing 3 logs (99.9%) of clonogenic lymphoma cells after a 2-hour incubation. The presence of human bone marrow inhibits the activity of the conjugate. However, full potency of TEC IgM-SAP immunotoxin is restored by adding 1 mM amantadine to the incubation medium. The reaction is highly specific and is inhibited by the presence of excess anti-mu-antibody or human serum. Clonal growth of other Burkitt's lymphoma cell lines is inhibited to a lesser extent by the immunotoxin. The presence of surface IgM on the different cell lines is directly correlated to target cell killing by TEC IgM-SAP. Isolation of Bjab 113 clones surviving treatment demonstrates that only a minority are truly resistant and that the others randomly escape the treatment. The highly potent and specific activity of this conjugate in the presence of bone marrow buffy coat and its exceptionally rapid onset of action make this conjugate a good candidate for the ex vivo elimination of neoplastic cells from the bone marrow of non-Hodgkin's lymphoma patients.

Antitumor activity of basic fibroblast growth factor-saporin mitotoxin in vitro and in vivo.

Many cancer cell lines express basic fibroblast growth factor (FGF) receptors, making them potential targets for the delivery of FGF-based cytotoxic compounds. To this end, we have investigated the antitumor activity of a novel mitotoxin, Fibroblast Growth Factor-saporin (FGF-SAP), a conjugate of FGF and the ribosome-inactivating protein, saporin. In vitro, FGF-SAP is cytotoxic for human melanoma, teratocarcinoma, and neuroblastoma cells expressing FGF-receptors. Mice treated with FGF-SAP i.v., on a variety of schedules, showed dramatic tumor growth inhibition with minimal toxicity. Thus, FGF-SAP appears to be a well-tolerated and potent antitumor agent. The potential of FGF-targeted cytotoxicity is discussed.

Immunotoxin-mediated inhibition of chronic lymphocytic leukemia cell proliferation in humans.

We evaluated the cytotoxicity of the immunotoxin OKT1-SAP on fresh B-chronic lymphocytic leukemia (B-CLL) cells from 31 consecutive patients. OKT1-SAP comprised the OKT1 (CD5) monoclonal antibody disulfide linked to saporin-6 (SAP) ribosome-inactivating protein from the plant Saponaria officinalis. The effect of OKT1-SAP on target CD5-positive B-CLL cells was estimated using an in vitro proliferation inhibition assay in which control or OKT1-SAP-treated B-CLL cells were induced to proliferate by sequential stimulation with insolubilized anti-C3b receptor CB04 (CD35) antibody and low molecular weight B-cell growth factor. In 90% of patients, OKT1-SAP specifically suppressed B-CLL cell proliferation in a dose-related manner (50% inhibitory concentration, 4.0-6.8 nM). Taken together the findings reported in this article provide information relevant to the clinical development of immunotoxins because: (a) the in vitro conditions under which B-CLL cell proliferation is inhibited by OKT1-SAP are achievable in vivo without nonspecific toxicity according to our previous toxicology and pharmacokinetics studies in primates; and (b) the B-CLL cell proliferation inhibition assay described here provides a basis for future comparative studies.

Schwann cells are removed from the spinal cord after effecting recovery from paraplegia.

Remyelination of the CNS is necessary to restore neural function in a number of demyelinating conditions. Schwann cells, the myelinating cells of the periphery, are candidates for this purpose because they have more robust regenerative properties than their central homologs, the oligodendrocytes. Although the ability of Schwann cells to remyelinate the CNS has been demonstrated, their capacity to enter the adult spinal cord in large numbers and effect functional recovery remains uncertain. We used cholera toxin B-subunit conjugated to saporin to demyelinate the rat lumbar spinal cord, remove macroglia, and produce paraplegia. After the removal of oligodendrocyte and astrocyte debris by invading macrophages, there was a spontaneous entry of Schwann cells into the spinal cord, along with axonal remyelination and concomitant functional recovery from paraplegia occurring within 75 d. The Schwann cells appeared to enter the dorsal funiculi via the dorsal root entry zone and the lateral funiculi via rootlets that had become adherent to the lateral spinal cord after the inflammation. In the following weeks, Schwann cell myelin surrounding central axons was progressively replaced by oligodendrocyte myelin without lapse in motor function. Our results show that endogenous Schwann cells can reverse a severe neurological deficit caused by CNS demyelination and enable later oligodendrocyte remyelination.

Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat.

Hypocretins (Hcrts) are recently discovered peptides linked to the human sleep disorder narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice also have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither electrolytic nor pharmacological lesions of this or any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. Hcrt neurons express the Hcrt receptor, and to facilitate lesioning these neurons, the endogenous ligand hypocretin-2/orexin B (Hcrt2) was conjugated to the ribosome-inactivating protein saporin (SAP). In vitro binding studies indicated specificity of the Hcrt2-SAP because it preferentially bound to Chinese hamster ovary cells containing the Hcrt/orexin receptor 2 (HcrtR2/OX(2)R) or the Hcrt/orexin receptor 1 (HcrtR1/OX(1)R) but not to Kirsten murine sarcoma virus transformed rat kidney epithelial (KNRK) cells stably transfected with the substance P (neurokinin-1) receptor. Administration of the toxin to the LH, in which the receptor is known to be present, eliminated some neurons (Hcrt, melanin-concentrating hormone, and adenosine deaminase-containing neurons) but not others (a-melanocyte-stimulating hormone), indicating specificity of the toxin in vivo. When the toxin was administered to the LH, rats had increased slow-wave sleep, rapid-eye movement (REM) sleep, and sleep-onset REM sleep periods. These behavioral changes were negatively correlated with the loss of Hcrt-containing neurons but not with the loss of adenosine deaminase-immunoreactive neurons. These findings indicate that damage to the LH that also causes a substantial loss of Hcrt neurons is likely to produce the multiple sleep disturbances that occur in narcolepsy.

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

The ability to selectively lesion mouse basal forebrain cholinergic neurons would permit experimental examination of interactions between cholinergic functional loss and genetic factors associated with neurodegenerative disease. We developed a selective toxin for mouse basal forebrain cholinergic neurons by conjugating saporin (SAP), a ribosome-inactivating protein, to a rat monoclonal antibody against the mouse p75 nerve growth factor (NGF) receptor (anti-murine-p75). The toxin proved effective and selective in vitro and in vivo. Intracerebroventricular injections of anti-murine-p75-SAP produced a dose-dependent loss of choline acetyltransferase (ChAT) activity in the hippocampus and neocortex without affecting glutamic acid decarboxylase (GAD) activity. Hippocampal ChAT depletions induced by the immunotoxin were consistently greater than neocortical depletions. Immunohistochemical analysis revealed a dose-dependent loss of cholinergic neurons in the medial septum (MS) but no marked loss of cholinergic neurons in the nucleus basalis magnocellularis after intracerebroventricular injection of the toxin. No loss of noncholinergic neurons in the MS was apparent, nor could we detect loss of noncholinergic cerebellar Purkinje cells, which also express p75. Behavioral analysis suggested a spatial learning deficit in anti-murine-p75-SAP-lesioned mice, based on a correlation between a loss of hippocampal ChAT activity and impairment in Morris water maze performance. Our results indicate that we have developed a specific cholinergic immunotoxin for mice. They also suggest possible functional differences in the mouse and rat cholinergic systems, which may be of particular significance in attempts to develop animal models of human diseases, such as Alzheimer's disease, which are associated with impaired cholinergic function.

Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.

Neurons in the rostroventromedial medulla (RVM) project to spinal loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a mechanism of chronic pain. This possibility and the phenotype of RVM cells that might underlie experimental neuropathic pain were investigated. Cells expressing mu-opioid receptors were targeted with a single microinjection of saporin conjugated to the mu-opioid agonist dermorphin; unconjugated saporin and dermorphin were used as controls. RVM dermorphin-saporin, but not dermorphin or saporin, significantly decreased cells expressing mu-opioid receptor transcript. RVM dermorphin, saporin, or dermorphin-saporin did not change baseline hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stimuli applied to the paw. RVM dermorphin or saporin did not alter SNL-induced experimental pain, and no pretreatment affected the responses of sham-operated groups. This protective effect of dermorphin-saporin against SNL-induced pain was blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with the mu-opioid receptor. RVM microinjection of dermorphin-saporin, but not of dermorphin or saporin, in animals previously undergoing SNL showed a time-related reversal of the SNL-induced experimental pain to preinjury baseline levels. Thus, loss of RVM mu receptor-expressing cells both prevents and reverses experimental neuropathic pain. The data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.

Selective elimination of fibroblasts from pancreatic islet monolayers by basic fibroblast growth factor-saporin mitotoxin.

Fibroblast proliferation regularly impedes the initiation and maintenance of pancreatic islet monolayers in culture. We recently characterized a specific cytotoxin to cells expressing the basic fibroblast growth factor receptor by conjugating the growth factor to saporin-6, a ribosome-inactivating protein. In contrast to untreated islets, isolated adult rat islets grown on a substrate prepared from bovine corneal endothelial cells and incubated with the mitotoxin at 10-nM concentration for 96 h were free of contaminating fibroblasts. Histological and functional studies revealed there was no damage to the islets. The results suggest that treatment of this cell type with basic fibroblast growth factor mitotoxins may be an important tool for culture of pure islets for physiological and clinical studies.

Molecular atherectomy for restenosis.

Receptors for basic (b) and acidic (a) fibroblast growth factor (FGF) are upregulated in activated smooth muscle cells. These cells, which proliferate in response to bFGF, can thus be killed by a conjugate of bFGF and the ribosome-inactivating enzyme, saporin (which, by itself, does not enter the cells). Quiescent smooth muscle cells and other cells that have few FGF receptors are not killed. In vivo, bFGF-saporin transiently inhibits smooth muscle cell proliferation and neointimal accumulation after balloon injury to the rat carotid artery. Delivery of saporin, diagnostic imaging agents, or antisense oligodeoxynucleotides might be made even more selective by linking these substances to antibodies against the extracellular domains of the putative FGF receptor isoform specific for activated smooth muscle cells.

Fibroblast growth factor in the hypothalamic-pituitary axis: differential expression of fibroblast growth factor-2 and a high affinity receptor.

In situ hybridization and immunohistochemistry were used to map gene expression and protein distribution of basic fibroblast growth factor (FGF-2) in the hypothalamic-pituitary system. Although the expression of FGF-2 mRNA in the pituitary is low, the protein is widely distributed in both its neural and anterior lobes. In the anterior lobe, immunoreactive (ir-) FGF-2 localizes to basement membranes and select endocrine cells. In the neural lobe, ir-FGF-2 is detected in basement membranes, pituicytes, and Herring bodies. Analyses of FGF high affinity receptor (FGFR) immunoreactivity in the anterior pituitary establishes a distribution of FGFR similar to that of FGF-2. In the neural lobe, ir-FGFR is associated with nerve fibers, pituicytes, and Herring bodies. Unlike FGF-2, the distribution of FGFR1 mRNA correlates well with the presence of the immunoreactive receptor. In the hypothalamus, magnocellular neurons of paraventricular and supraoptic nuclei contain ir-FGF-2 and ir-FGFR. In the median eminence, ir-FGF-2 and ir-FGFR is associated with fibers, glial, and endothelial cells. Ependymal and subependymal cells lining the third ventricle also show high levels of ir-FGF-2 and ir-FGFR and mRNAs. Overall, there is a specific and selective distribution of FGF-2 and its high affinity receptor(s) in the hypothalamo-pituitary axis. This localization lead us to postulate a role in neurohypophyseal functions, possibly water balance.

Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.

CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.

Functional impact of attachment and purification in the short term culture of human pancreatic islets.

We have evaluated the effects on islet function of several manipulations of the substrate and tissue culture conditions in the short term culture of human islets. Specifically, we have studied the influence of several matrices, additions to the medium, and the use of basic fibroblast growth factor (FGF)-saporin mitotoxins to eliminate fibroblastoid cells from the cultures. The human islets were obtained from the Human Islet Transplant Center at Washington University Medical Center (St. Louis, MO). Substrates used to facilitate islet attachment were poly-L-lysine, gelatin, Matrigel, collagen, and bovine corneal endothelial cell matrix. RPMI-1640 medium was supplemented with either 22.2 mM glucose or 10 micrograms/mL human insulin. FGF-saporin mitotoxin was used at a concentration of 10 nM. The greatest improvement in islet cell function in either static or stimulated situations was obtained when we used bovine corneal endothelial cell matrix as the matrix, supplemented the medium with a high concentration of glucose or insulin, and eliminated fibroblast-like cells by exposing the cultures to basic FGF-saporin mitotoxin. The conditions described in this report could greatly improve the culture of human islets for use in clinical and laboratory research.

Plasmid pFCE4: a new system of Escherichia coli expression-modification vectors.

Two versatile expression-modification vectors were obtained by inserting the origin of replication (ori) of phage f1 into the expression vector pOTS. The resulting plasmids produce large amounts of coding or noncoding ssDNA (depending on ori orientation in pFCE4+ and pFCE4-) and excrete it into the medium as virus-like particles following infection with phage f1. These features make them suitable for dideoxy chain termination sequencing, oligonucleotide directed mutagenesis and gene expression without further manipulations. The human IFN alpha-2 gene, lacking the codon for the first amino acid, cysteine, was efficiently expressed by these vectors.

Expression of the human apolipoprotein AI gene fused to the E. coli gene for beta-galactosidase.

The human apoAI gene was expressed in E. coli by in-frame fusion to a modified beta-galactosidase gene present in plasmid pUR291. The fused beta-galactosidase-apoAI gene product was expressed at a high level and was recognized by an anti-human apoAI antiserum. Besides the fused protein, at least one degradation product having an Mr similar to that of beta-galactosidase was present in high amounts in bacterial extracts. These results and those of a pulse-chase experiment indicate that degradation took place only in the apoAI moiety of the chimeric protein.

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation.

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

Retrograde degeneration and colchicine protection of basal forebrain cholinergic neurons following hippocampal injections of an immunotoxin against the P75 nerve growth factor receptor.

Intracerebroventricular injection of 192 IgG antibody against the p75LNGFR rat low affinity nerve growth factor receptor conjugated with saporin, a ribosome inactivating protein, has been shown to destroy the p75LNGFR-expressing cholinergic neurons of the basal forebrain. We injected this immunotoxin into the hippocampus and studied its retrograde effect upon the cholinergic neurons of the medial septum and the vertical limb of the diagonal band of Broca. Seven days after injection, there was a nearly total depletion of cholinergic axons within the hippocampus. This depletion was associated with a marked and significant decrease in the number of cholinergic neurons of the ipsilateral medial septum and the vertical limb of the diagonal band of Broca. At longer survival times, these changes were more pronounced. Parvalbumin-positive, GABAergic neurons within the same areas of the basal forebrain were not affected by immunotoxin injections. Injections of saporin alone had no effect upon cholinergic neurons. Simultaneous injection of colchicine with the immunotoxin resulted in a significant reduction of retrograde degeneration of cholinergic neurons and relative preservation of hippocampal cholinergic axons. These observations suggest that 192 IgG-saporin is transported retrogradely from the hippocampus to the cholinergic neurons in the medial septum and the vertical limb of the diagonal band of Broca and provide a model for retrograde degeneration of basal forebrain cholinergic neurons following cortically based toxic-pathologic processes.

192 immunoglobulin G-saporin produces graded behavioral and biochemical changes accompanying the loss of cholinergic neurons of the basal forebrain and cerebellar Purkinje cells.

Immunolesions of the cholinergic basal forebrain were produced in rats using various intraventricular doses of the immunotoxin 192 immunoglobulin G-saporin: 0.34, 1.34, 2.0, 2.7 and 4.0 micrograms/rat. A battery of behavioral tests, chosen on the basis of reported sensitivity to conventional medial septal or nucleus basalis lesions, was administered. Dose-dependent impairments were found in acquisition, spatial acuity and working memory in the water maze. Dose-dependent hyperactivity in the open field and in swimming speed was observed. The highest dose group (4.0 micrograms) exhibited motoric disturbances which were particularly apparent in swimming and in clinging to an inclined screen. Response and habituation to acoustic startle were diminished in the three higher dose groups. Histological results from acetylcholinesterase and low-affinity nerve growth factor receptor staining showed that the lesion was selective for cholinergic neurons bearing p75 nerve growth factor receptors in the basal forebrain nuclei. However, some Purkinje cells in the superficial layers of the cerebellum were also destroyed at the higher doses of immunotoxin. The activity of choline acetyltransferase, used as a marker of cholinergic deafferentation in regions innervated by the basal forebrain nuclei, was decreased with increasing doses to a plateau level of about 90% (average depletion) for the two highest dose groups. These two groups were the only ones to exhibit consistent and severe behavioral impairments on all behavioral tests performed. Thus, for a relatively selective cholinergic basal forebrain lesion, almost a 90% reduction in choline acetyltransferase activity is needed to produce substantial behavioral deficits. It appears that either a considerable safety factor exists or robust compensatory mechanisms can ameliorate behavioral deficits from a major, but incomplete loss of cholinergic basal forebrain innervation.

Behavioural, histochemical and biochemical consequences of selective immunolesions in discrete regions of the basal forebrain cholinergic system.

The effectiveness of a recently developed immunotoxin, 192 IgG-saporin, was evaluated for making selective lesions of subgroups of basal forebrain cholinergic neurons. Following a pilot series of injections into the nucleus basalis magnocellularis to establish the effective dose for intraparenchymal lesions, separate groups of rats received injections of the immunotoxin into the septum, into the diagonal band of Broca or into the nucleus basalis magnocellularis. The lesions produced extensive and effective loss of cholinergic neurons in the discrete areas of the basal forebrain, as identified by loss of cells staining for acetylcholinesterase and p75NGFr, with a parallel loss of acetylcholinesterase staining and choline acetyltransferase activity in the target areas associated with each injection site in the dorsolateral neocortex, cingulate cortex and hippocampus. The selectivity of the lesion for cholinergic neurons was supported by the lack of gliosis and sparing of small to medium-sized cells at the site of injection of the toxin, including the glutamate decarboxylase immunoreactive cells that contribute to the septohippocampal projection. In spite of the extensive disturbance in the cholinergic innervation of the neocortex and hippocampus, immunotoxin lesions produced no detectable deficit in the Morris water maze task in any of the lesion sites within the basal forebrain. By contrast small but significant deficits were seen on tests of nocturnal activity (septal and nucleus basalis magnocellularis lesions), open field activity (septal and diagonal band lesions), passive avoidance (nucleus basalis magnocellularis lesions) and delayed non-matching to position (septal lesions). The results indicate that the 192 IgG-saporin provides a powerful tool for making effective lesions of the basal forebrain cholinergic neurons, and that the behavioural sequelae of such lesions warrant further detailed investigation.