Phospholipase A2 activating protein induces tumor regression.

There has been increasing interest in attempts to harness the body's normal inflammatory response mediated through the eicosanoid pathway to treat tumors. Accumulating data indicate that the growth of several different cancers is modulated by a group of pro-inflammatory bioactive lipids, the best known of which are the eicosanoids. Eicosanoid pathway constituents modulate cell function in several important ways, and an agent that activates PLA(2) and up-regulates LTB(4) levels could be expected to be an effective cytotoxic tumor agent, especially if it stimulated NK cells. PLAP is a 28-kDa polypeptide that is a member of the WD-repeat protein, G-protein-transducin superfamily. The pro-inflammatory properties of PLAP have been elucidated using a number of different approaches. PLAP has been found in inflamed tissues and synovial fluid from patients with rheumatoid arthritis. Based on knowledge of PLAP as a pro-inflammatory agent, its capacity to modulate the immune response and the role of the inflammatory and immune responses in immune surveillance, the role of PLAP in cancer therapy was explored. Significant tumor regression was observed 72 hours following a single treatment with PLAP in an animal air pouch model of glioma. PEG-PLAP treatment increased the life expectancy of animals with Lewis lung cancer, and in preliminary studies in MTVL breast tumors in mice, PLAP treatment resulted in a similar increase in life expectancy. These findings suggest that PLAP holds promise as a potential therapy for cancer, and warrants further study.

Cholera toxin induces synthesis of phospholipase A2-activating protein.

The mechanism of cholera toxin (CT)-stimulated arachidonate metabolism was evaluated. CT caused rapid in vitro synthesis of prostaglandin E2 (PGE2) in murine smooth muscle-like cells (BC3H1), reaching maximal levels within 3 to 4 min. In comparison, cyclic AMP (cAMP) levels were unchanged, and addition of dibutyryl cAMP did not affect PGE2 synthesis. CT-induced PGE2 synthesis was prevented by actinomycin D or cycloheximide, indicating a need for de novo protein synthesis. Northern blot analysis of total RNA from BC3H1 cells revealed that exposure to CT resulted in an increase in abundance of mRNA encoding phospholipase A2 (PLA2)-activating protein (PLAP). PLAP is a regulatory protein that increases the enzymatic activity of cellular PLA(2), which in turn causes increased hydrolysis of arachidonate from membrane phospholipids. Furthermore, CT evoked the accumulation of PLAP mRNA in J774 (murine monocyte/macrophage) and Caco-2 (human intestinal epithelial) cells in vitro, but the responses were more delayed than that of BC3H1 cells. A protein band of approximately 35 kDa, which corresponded to the size of PLAP, was observed in sodium dodecyl sulfate extracts of Caco-2 cells by Western blot (immunoblot) analysis using affinity-purified antibodies to PLAP synthetic peptides. Synthesis of PLAP protein was increased after 2 h of exposure to CT. Exposure of mouse intestinal loops to either CT or live Salmonella typhimurium for 3 h increased mucosal PLAP mRNA levels. The role of PLAP in CT-induced PGE2 synthesis provides an attractive explanation for the reported suppression of CT-induced intestinal secretion by inhibitors of protein synthesis.

Phospholipase A2-mediated inflammation induces regression of malignant gliomas.

An ideal form of cancer therapy is the harnessing of innate immunity to eradicate spontaneously arising clones of malignant cells. To date, attempts to develop effective immunotherapies have met with limited success. Prostaglandins and leukotrienes, collectively known as eicosanoids, are important mediators of immune and inflammatory responses. Harnessing these compounds could be a method to treat cancers. Eicosanoids are formed after cleavage of fatty acids from phospholipids by phospholipase enzymes. We have previously described, characterized and cloned a naturally occurring mammalian activator of phospholipase A2. Injection of a 24 amino acid peptide from this phospholipase A2 activating protein (PLAP), resulted in induction of an acute inflammatory response, and a concomitant regression of gliomas in rats. Administration of 500 micrograms of this protein resulted in a 50% decrease of the tumor mass within 72 h. Tumor regression coincided with a greater than twenty-fold increase in levels of prostaglandin E2(PGE2) and leukotriene B4(LTB4), and a marked infiltration of natural killer(NK) cells. These data suggest that activation of phospholipase A2 and modulation of the eicosanoid biosynthetic pathway may provide a novel therapeutic strategy for the successful treatment of malignant tumors of the nervous system.

Clinical assessment of the 1987 American College of Rheumatology criteria for rheumatoid arthritis.

The 1987 American College of Rheumatology (ACR) criteria for the classification of rheumatoid arthritis (RA) were clinically assessed. These criteria do not include findings of synovial fluid (SF) analysis and require no exclusion criteria. We have studied sequential patients with arthritis seen in four rheumatology centers in the Philadelphia area. Classifications by the ACR criteria were compared with our clinical diagnoses. Two hundred ninety eight patients were evaluated, 113 with RA and 185 with other diagnoses. Classifications as RA by the ACR criteria corresponded to our clinical diagnosis in 95% of the cases, corroborating the high sensitivity previously reported. However, we found a lower specificity (73%) than that reported (89%). False positive classifications as RA were found in 71% of patients with psoriatic arthritis, 48% of patients with SLE, and 31% of patients with gout. The specificity could be improved to 89% by excluding disorders with obvious distinguishing extraarticular features such as psoriasis or by SF findings of monosodium urate crystals. Awareness of these possible sources of confusion will further increase the teaching and epidemiologic value of these useful simplified criteria.

Phospholipase A2-activating protein induces the synthesis of IL-1 and TNF in human monocytes.

Phospholipase A2-activating protein (PLAP) is an important mediator of eicosanoid generation. PLAP can also be found in high concentrations in synovial fluid from patients with rheumatoid arthritis, and injection of PLAP into animal joints results in an inflammatory, rheumatoid-like lesion. We have demonstrated previously that TNF-alpha and IL-1 beta stimulate formation of PLAP before phospholipase A2 (PLA2) enzyme activation and production of eicosanoids. To further explore the mechanisms found in the inflammatory response, we examined the ability of PLAP to stimulate release of TNF and IL-1 from human peripheral blood monocytes. TNF and IL-1 protein levels were measured by ELISA, and IL-1 and TNF mRNA were determined by Northern blotting. PLAP, PLAP peptide, and melittin, a bee venom PLA2 activator with homology with PLAP, all increased IL-1 and TNF production in a time- and dose-dependent manner. Heat-denatured PLAP and actin (an irrelevant protein) failed to exert this effect. PLAP stimulation of TNF and IL-1 could be enhanced with co-treatment of cells with free fatty acids, such as arachidonic or linoleic acid, but it was not blocked completely by PLA2 inhibitors. These results demonstrate not only that synthesis of PLAP can be stimulated by cytokines, but also that PLAP may regulate cytokine synthesis and thus perpetuate an immune or inflammatory response.

Activation of mechanonociceptors by pro-inflammatory peptides melittin and PLAP peptide.

Studies were conducted to characterize the chemical reactivity of A and C fiber mechanical and mechanoheat nociceptors that innervate the goat palatal mucosa. In mechanical nociceptors, no significant chemical reactivity to either serotonin, bradykinin, prostaglandin E1, or prostaglandin E2 was observed, regardless of whether substances were injected singly or in spaced sequential combinations. Weak reactivity was observed in mechanoheat nociceptors. In contrast, both mechanical and mechanoheat nociceptors were activated by novel proinflammatory peptides. Nineteen of 30 mechanonociceptors and 12 of 13 mechanoheat nociceptors were activated by the insect venom peptide, melittin or its endogenous mammalian homologue PLAP (phospholipase A2 activating protein) peptide. Low threshold mechanoreceptors were never activated by melittin or PLAP peptide.

Phospholipase A2 activity in human neutrophils. Stimulation by lipopolysaccharide and possible involvement in priming for an enhanced respiratory burst.

Exposure to LPS, platelet-activating factor, certain cytokines, and other agents can prime human neutrophils for an increased release of superoxide anion (O2-) in response to stimuli. Previous work with LPS has suggested that priming may involve alterations in signal transduction pathways related to the release of O2-. Products derived from the enzymatic activity of phospholipase A2 (PLA2) on membrane phospholipids reportedly activate certain of these signaling events. Thus, PLA2 could play a regulatory role in the release of O2- by neutrophils. We examined this possibility by studying the effect of LPS on neutrophil PLA2 activity. Exposure to LPS triggered a fivefold increase in activity of an endogenous PLA2; control cells incubated under identical conditions without LPS showed no increase. Neutrophil-associated PLA2 activity increased 5 to 10 min after the addition of LPS to the cells and preceded the emergence of the primed state. Quinacrine and p-bromophenacylbromide, inhibitors of PLA2, blocked LPS priming but not the baseline O2- release from unprimed cells. The LPS-provoked increase in PLA2 activity resulted in release of oleic acid (38 +/- 4% above baseline) but not arachidonic, linoleic, or palmitic acid. In contrast, ionomycin resulted in significant release of all four fatty acids. The addition of exogenous PLA2 to neutrophils primed them. Priming was rapid and was 80 +/- 5% complete within 3 min. Thus, LPS and perhaps other agents may mediate their effects on O2- release at least in part by triggering PLA2 activity. PLA2 activation may play a role in regulating production and release of O2- by the human neutrophil.

Phospholipase A2 and arthritis.

Over the last 30 years, interest in PLA2 has grown beyond its enzymatic capacity to cleave phospholipids. It has been recognized as the rate-limiting step in the release of arachidonic acid and subsequent formation of prostaglandins, leukotrienes, and other bioactive lipids. Subsequently, PLA2 has not only been found to be present in high concentrations in inflammatory arthritis, but also to induce inflammation when injected into animals. At the same time, investigators into mechanisms of signal transduction demonstrated that a variety of cytokines including IL-1 and TNF, which are found in high concentrations in synovial fluid from patients with RA, stimulate PLA2 activity. These investigations demonstrated further the central role for PLA2 in inflammatory events, especially inflammatory arthritis. Numerous other PLA2 proteins, in addition to the low molecular weight synovial fluid/platelet enzyme, also have been characterized. Their clinical role in arthritis is yet to be elucidated. Human proteins which either inhibit or stimulate PLA2 have also been identified, characterized, and cloned. More recently, exciting investigations, primarily from biotechnology and pharmaceutical companies, into inhibitors of PLA2 have been reported. New PLA2-regulating compounds, which will hopefully move from the laboratory and through clinical trials and then be used to treat patients with arthritis, are on the horizon.

Responses of purified phospholipases A2 to phospholipase A2 activating protein (PLAP) and melittin.

The role of the phospholipase A2 (PLA2) stimulating protein PLAP in the regulation of PLA2 activity was assessed by determination of the effects of PLAP on two purified PLA2s. An approx. 14 kDa enzyme was purified from mouse thymoma cells, EL-4 cells, by cation ion exchange HPLC and immunoaffinity HPLC (with antiserum to the N-terminal sequence of an inflammatory exudate PLA2). An approx. 110 kDa enzyme was purified from mouse mammary carcinoma derived cells by sequential hydrophobic, anion exchange, hydroxyapatite and gel filtration HPLC. Neither PLAP nor melittin, an immunologically related PLA2 stimulating peptide from bee venom, increased the activity of the high molecular weight enzyme. In contrast, there was more than a 20-fold stimulation of the low molecular weight PLA2 by PLAP and an approx. 5-fold stimulation by melittin. The stimulation of enzyme activity by PLAP was observed at a protein to phospholipid ratio of 1:10(6) while the ratio of melittin to phospholipid was 1:3. Thus, PLAP mediated stimulation of PLA2 activity may include an interaction between PLAP and the enzyme, in contrast to melittin stimulation, which involves interactions between melittin and phospholipid.

Regulation of synovial cell growth: basic fibroblast growth factor synergizes with interleukin 1 beta stimulating phospholipase A2 enzyme activity, phospholipase A2 activating protein production and release of prostaglandin E2 by rheumatoid arthritis synovial cells in culture.

Cytokines have been implicated in the regulation of eicosanoid synthesis and synovial cell proliferation. To further define these mechanisms, we have compared the effects of basic fibroblast growth factor and platelet-derived growth factor on cell growth, prostaglandin E2 (PGE2) production and phospholipase A2 enzyme activity in long-term cultures of synovial cells from rheumatoid arthritis (RA) patients capable of proliferating in serum-free medium. Compared with serum-free medium alone, RA synovial cell growth was significantly enhanced by adding either basic fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF) to the culture medium. Growing RA synovial cells for 14 days in serum-free medium plus bFGF caused them to spontaneously release significant amounts of PGE2, an effect not seen if cells were grown in serum-free medium alone, or serum-free medium plus PDGF. Enhanced release of PGE2 occurred when arachidonic acid was added to bFGF but not PDGF-treated RA synovial cells, suggesting that bFGF increased cyclooxygenase enzyme activity in these cells. Moreover, phospholipase A2 (PLA2) enzyme activity was found to be significantly greater in RA synovial cells grown for 14 days in serum-free medium containing bFGF alone, or bFGF plus interleukin 1 beta (IL-1 beta) compared with cells grown in either serum-free medium alone, or serum-free medium plus PDGF. Similarly, bFGF plus IL-1 beta-stimulated release of PLA2 activating protein, a novel mammalian phospholipase stimulator found in high concentrations in RA synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

IL-1 increases phospholipase A2 activity, expression of phospholipase A2-activating protein, and release of linoleic acid from the murine T helper cell line EL-4.

The early events in IL-1-mediated activation of T cells were investigated in the murine T cell line, EL-4. Treatment of EL-4 cells with human rIL-1 beta resulted in a rapid increase in phospholipase A2 (PLA2) activity. PLA2 activity increased approximately fivefold within 4 min after exposure to IL-1. Synthesis of the phospholipase A2- activating protein (PLAP) and its mRNA were also increased within 4 min of IL-1 treatment and preceded the increase in PLA2 enzyme activity. The increases in PLA2 activity and PLAP protein and mRNA levels were all transient and declined to baseline within 10 min after the addition of IL-1. The changes in levels of PLAP as a function of time after IL-1 treatment were consistent with PLAP playing an important role in the regulation of PLA2 activity in this system. The consequence of the elevated PLA2 activity was examined by analysis of the fatty acids released from IL-1-treated cells. There was a 20-fold increase in the release of radioactivity from [14C]-linoleic acid labeled cells whereas there was very little change in the release of radioactivity from [14C]-arachidonic acid labeled cells in response to the addition of IL-1. The radioactivity released from [14C]-linoleic acid labeled cells was analyzed by HPLC; no conversion of radiolabeled linoleic into arachidonic acid was observed. In EL-4 cells, IL-1 potentiates PMA-mediated release of IL-2 at suboptimal concentrations of PMA. Linoleic acid also augmented PMA-induced IL-2 release from the EL-4 cells. This fatty acid was more than 10 times more effective than arachidonic acid in this regard. Furthermore, the addition of exogenous PLAP to EL-4 cells could substitute for IL-1 in the stimulation of IL-2 release. These results suggest that the IL-1 effects on T cells may be mediated at least in part through increased PLA2 activity due to increased synthesis of PLAP. Furthermore, the release of the unsaturated fatty acid linoleic acid or its metabolites may be of functional importance in IL-1-mediated IL-2 production by EL-4 cells.

Cloning of a phospholipase A2-activating protein.

Recently we have described the isolation and biochemical characterization of a phospholipase A2-activating protein (PLAP). We have cloned this protein and found it to be expressed as a 2.5-kilobase mRNA. The steady-state levels of PLAP mRNA are induced in smooth muscle and endothelial cells following treatment with leukotriene D4. The increased message levels coincide with increased amounts of PLAP. Synthetic antisense DNA was used to block the synthesis of PLAP and this treatment effectively blocked the activation of phospholipase A2 and the increased generation of prostanoids in smooth muscle and endothelial cells treated with leukotriene D4.

Human extracellular recombinant phospholipase A2 induces an inflammatory response in rabbit joints.

Phospholipase A2 (PLA2) enzymes hydrolyze membrane phospholipids liberating fatty acid and lysophospholipid. This event is thought to be the rate-limiting step in the generation of the lipid proinflammatory mediators, the eicosanoids and possibly platelet-activating factor. For this reason, extracellular forms of PLA2 have been postulated to be a component of the inflammatory cascade in certain biologic settings. In the synovial fluid of patients with inflammatory arthritides, substantial amounts of PLA2 activity have been found, and subsequently, one enzyme was purified, cloned, and expressed. Here we show that the pure recombinant enzyme free of any proinflammatory contaminants elicits a dramatic inflammatory, arthritogenic response when injected into the joint space of healthy rabbits. Within 24 h, extensive leukocyte infiltration and hyperplasia of the synovial lining cells were observed, and prostaglandin production in the joint space increased. In comparison, pancreatic PLA2(2) had little activity in this system, whereas a very inflammatory cobra venom enzyme was intermediate in its effects. In view of the presence of the enzyme in inflamed joints, the fact that its synthesis and secretion can be induced by proinflammatory cytokines and its proinflammatory biologic activity, we suggest that synovial PLA2 plays an exacerbating role in acute episodes in chronic inflammatory conditions such as rheumatoid arthritis.

Yeast mitochondria (Saccharomyces cerevisiae) contain Ca(2+)-independent phospholipase A1 and A2 activities: effect of respiratory state.

We demonstrate that both phospholipase A1 and phospholipase A2 are associated with isolated yeast mitochondria (Saccharomyces cerevisiae). Activity assays indicate that, unlike most other mitochondrial phospholipases A, the yeast enzymes are Ca(2+)-independent with acidic (pH 4-5) as well as alkaline (pH 8-9) pH optima. Data obtained with mitochondria isolated from either fermenting or respiring cells, and initial observations with a petite strain, strongly suggest that a phospholipase A2 with an acidic pH optimum functions in the in vivo adaptation and maintenance of mitochondrial membranes required for respiration.

Identification and isolation of a phospholipase A2 activating protein in human rheumatoid arthritis synovial fluid: induction of eicosanoid synthesis and an inflammatory response in joints injected in vivo.

Eicosanoids are important mediators of the destructive arthropathy observed in rheumatoid arthritis. The rate-limiting step in the eicosanoid synthesis pathway is the availability of free arachidonic acid. The phospholipase enzymes release arachidonic acid from membrane phospholipids and thus play an important role in the regulation of eicosanoid production. We have previously demonstrated enhanced phospholipase A2 and C enzyme activities in cells from patients with rheumatoid arthritis and have also described a phospholipase A2 activating protein (PLAP) in mammalian cell lines. In an attempt to determine the biochemical basis of enhanced phospholipase A2 activity found in patients with inflammatory joint disease, we examined synovial fluid from patients with rheumatoid arthritis for PLAP. To determine whether PLAP was specific for rheumatoid disease, we assayed specimens from patients with other arthropathies. Histologic examination of rheumatoid joint tissue, with the use of immunohistochemical techniques, demonstrated high concentration of PLAP in monocytes, macrophages, chondrocytes, vascular smooth muscle, and endothelial cells. Human PLAP could be biochemically isolated from synovial fluid from patients with rheumatoid arthritis and was found to be similar to PLAP previously isolated from murine and bovine sources. To determine whether PLAP could directly mediate any aspect of inflammatory disease, purified PLAP was injected into rabbit knee joints. This resulted in an acute inflammatory arthritis with synovial cell proliferation and synovial fluid leukocytosis. Purified PLAP also induced eicosanoid formation both in vivo and in vitro. With enzyme-linked immunosorbent assays, we found more PLAP in synovial fluid specimens from patients with rheumatoid arthritis compared with samples from patients with other inflammatory arthropathies as well as osteoarthritis, a noninflammatory arthropathy. These data suggest that PLAP may be responsible, at least in part, for some aspects of the destructive inflammatory arthropathy that is observed in patients with rheumatoid arthritis.

Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein.

Eicosanoids are important mediators of the inflammatory response to monosodium urate crystals (MSUC) that results in gout. Phospholipase enzymes cleave fatty acids from membrane phospholipids, and this is thought to be the rate-limiting step in eicosanoid production. To understand better the mechanism of eicosanoid production in this disease, we stimulated human peripheral blood neutrophils and monocytes with MSUC and measured phospholipase enzyme activities. MSUC stimulated both intracellular and secretory phospholipase A2 enzyme activities in a time and concentration-dependent manner. Specificity was observed, as phospholipase C activities were not affected. Pretreatment with colchicine, but not aspirin, indomethacin, allopurinol, or islet activating protein, abrogated the enhanced phospholipase A2 activities. We have recently isolated and characterized a phospholipase A2 activating protein termed PLAP from synovial fluid from patients with rheumatoid arthritis, and from murine and bovine cell lines. PLAP was detected in gouty synovial fluid by immunodot blotting and ELISA assays and expressed the same characteristics as PLAP identified from other sources. To examine the role of PLAP in MSUC-induced phospholipase A2 stimulation, we treated cells with MSUC and observed an increase in immunoreactive PLAP. This response also could be blunted by colchicine, but not other drugs. Both phospholipase A2 and PLAP induced production by human monocytes of PGE2 and leukotriene B4 by neutrophils. These findings suggest that phospholipase A2 activation in response to MSUC requires an intact microtubule structure, and that phospholipase A2 and PLAP may be important modulators of at least a portion of the gouty inflammatory response.

Acute rheumatologic disorders in the elderly.

A variety of rheumatologic disorders affect the elderly. Some of these problems are seen almost exclusively in the elderly, such as temporal arteritis and pseudogout. Because of underlying chronic diseases, these patients are also at increased risk for joint infection and resultant sepsis. Evaluation of synovial fluid from the inflamed joint is important. Light microscopy evaluation with a red polarizing compensator can help diagnose crystal-mediated disease, such as gout or pseudogout. Examination of Gram stains can help diagnose infectious arthritis. Thus, appropriate processing of synovial fluid is mandatory for the diagnosis of many rheumatologic disorders that occur in the elderly. A variety of metabolic disorders are associated with pseudogout and should be searched for on laboratory evaluation. Appropriate laboratory evaluation and follow-up following the acute episode are important in the care of these patients. For example, temporal arteritis with resultant blindness is a feared disorder in the elderly. Transient blindness, headaches, jaw claudication, and an elevated Westergren sedimentation rate suggest this diagnosis. Aches and pain in the neck and shoulder area, especially in the morning, are typical of polymyalgia rheumatica. Polymyalgia rheumatica may also be a symptom of temporal arteritis.

Peripheral arthrocentesis in the work-up of acute low back pain.

Involvement of the axial skeleton in acute gouty arthritis has rarely been reported. Without the presence of peripheral tophi or history of gout, this diagnosis is often not considered. A patient is described with acute low back pain and for whom a diagnosis of acute axial gout was suggested after arthrocentesis of an asymptomatic peripheral joint yielded monosodium urate crystals. Treatment with colchicine led to prompt resolution of the gouty flare. Demonstration of urate crystals in this manner may encourage the clinician to attempt a trial therapy for acute gout, or at least to add gout to the differential diagnosis of acute back pain.

Leukotriene B4 modulates phospholipid methylation and chemotaxis in human polymorphonuclear leukocytes.

Formation of phosphatidylcholine from phosphatidylethanolamine via the S-adenosylmethionine (AdoMet) pathway has been shown to be required for signal transduction of receptor-ligand interactions in a variety of cells. These interactions result in the remodeling of phospholipid pools and phospholipase activation. To extend these observations and to explore the role of the phosphatidylcholine synthesis pathway in transduction of the leukotriene B4 (LTB4) receptor-ligand response, we examined phospholipid methylation in human polymorphonuclear leukocytes (PMN) following stimulation by LTB4, a potent chemotactic agent that is a metabolite of arachidonic acid. At early time points (approximately 3-10 min), formation of methylated phospholipids was enhanced following LTB4 stimulation. The LTB4 analogs 6-trans LTB4 as well as LTB4 epimers induced less methylation compared with LTB4, and the potencies of these analogs in inducing methylation correlated with their diminished ability to induce chemotaxis. Furthermore, the ability of these agonists to induce methylation also correlated with the binding affinity of these agents to the LTB4 receptors on these cells. Synthesis of phosphatidylcholine by the choline transferase pathway was not affected by LTB4. Inhibition of the AdoMet reaction with 3- deazaadenosine, L-homocysteine homolactone, or erythro-9-[2-hydroxy-3-nonyl] adenine (EHNA) abrogated LTB4-induced phospholipid methylation and the chemotactic response. The potencies of these inhibitors in blocking phospholipid methylation also correlated with their ability to abrogate the LTB4-induced chemotactic response. These data suggest that phospholipid methylation and phospholipase activation play an important role in transduction of the LTB4 receptor-ligand interaction in PMN, which results in chemotaxis.