Increased MAO-A Activity Promotes Progression of Pulmonary Arterial Hypertension.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses.

Adoptive T-cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, which introduces antigen-specific T-cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T-cell repertoires. Studies have suggested that use of higher-affinity TCRs against class I major histocompatibility complex antigens could drive the activity of both CD4(+) and CD8(+) T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here, we describe a high-throughput platform of 'reverse biochemistry' whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes (TILs) or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8(+) T cells expressing the highest-affinity TCR variants were deleted in both the TIL population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4(+) T cells in the tumor, suggesting they had a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR-binding properties that promote peripheral T-cell survival and tumor elimination.

Ag-doped manganite nanoparticles: new materials for temperature-controlled medical hyperthermia.

The purpose of this study was to introduce newly synthesized nanomaterials as an alternative to superparamagnetic ironoxide based particles (SPIO) and thus to launch a new platform for highly controllable hyperthermia cancer therapy and imaging. The new material that forms the basis for this article is lanthanum manganite particles with silver ions inserted into the perovskite lattice: La(1-x)Ag(x)MnO(3+delta). Adjusting the silver doping level, it is possible to control the Curie temperature (T(c)) in the hyperthermia range of interest (41-44 degrees C). A new class of nanoparticles based on silver-doped manganites La(1-x)Ag(x)MnO(3+delta) is suggested. New nanoparticles are stable, and their properties were not affected by the typical ambient conditions in the living tissue. It is possible to monitor the particle uptake and retention by MRI. When these particles are placed into an alternating magnetic field, their temperature increases to the definite value near T(c) and then remains constant if the magnetic field is maintained. During the hyperthermia procedure, the temperature can be restricted, thereby preventing the necrosis of normal tissue. A new class of nanoparticles based on silver-doped manganites La(1-x)Ag(x)MnO(3+delta) was suggested. Ag-doped perovskite manganites particles clearly demonstrated the effect of adjustable Curie temperature necessary for highly controllable cellular hyperthermia. The magnetic relaxation properties of the particles are comparable with that of SPIO, and so we were able to monitor the particle movement and retention by MRI. Thus, the new material combines the MRI contrast enhancement capability with targeted hyperthermia treatment.

An Ultrahigh Resolution SPECT System for I-125 Mouse Brain Imaging Studies.

This paper presents some initial experimental results obtained with a dual-head prototype single photon emission microscope system (SPEM) that is dedicated to mouse brain studies using I-125 labeled radiotracers. In particular, this system will be used for in vivo tacking of radiolabeled T cells in mouse brain. This system is based on the use of the intensified electron multiplying charge-coupled device (I-EMCCD) camera that offers the combination of an excellent intrinsic spatial resolution, a good signal-to-noise ratio, a large active area and a reasonable detection efficiency over an energy range between 27-140keV. In this study, the dual-head SPEM system was evaluated using both resolution phantoms and a mouse with locally injected T cells labelled with I-125. It was demonstrated that for a relatively concentrated source object, the current dual-head SPEM system is capable of visualizing the tiny amount of radioactivity (~12 nCi) carried by a very small number (<1000) of T cells. The current SPEM system design allows four or six camera heads to be installed in a stationary system configuration that offers a doubled or tripled sensitivity at a spatial resolution similar to that obtained with the dualhead system. This development would provide a powerful tool for in vivo and non-invasive tracking of radiolabeled T cells in mouse brain and potentially for other rodent brain imaging studies.

IL-12 treatment of endogenously arising murine brain tumors.

A number of recent studies have indicated that T cells can be stimulated to attack transplanted brain tumors in rodent models. As IL-12 has been shown to activate cytotoxic T cell responses, we tested the idea that it might stimulate a T cell response against endogenous brain tumors that arise in SV40 large T Ag transgenic mice (SV11). SV11 mice develop tumors of the choroid plexus, a specialization of the ependymal lining of the brain ventricles. They are a particularly relevant model of human disease, because they are immunocompetent but immunologically tolerant of the tumors. SV11 mice were treated with recombinant murine IL-12 for 10 days. Tumors grew more slowly than in control treated mice, and in some cases were reduced in size, as assessed by magnetic resonance imaging before and after treatment. At the end of treatment, tumors, but not brain parenchyma, exhibited extensive infiltration of activated CD8(+) and CD4(+) T cells. Tumors also showed a reduction in vascular density. Mice treated with IL-12 lived significantly longer than control mice. Tumors that progressed were nearly devoid of T cells, indicating that the T cell response was not sustained. In addition, some mice that had a substantial tumor burden at the beginning of treatment displayed evidence of immunosuppression, which might be related to TGF-ss2 detected in tumors. We conclude that IL-12 treatment can initiate an anti-tumor response even against endogenously arising brain tumors, but factors that will allow a sustained and more effective anti-tumor response need to be determined.

Bispecific agents target endogenous murine T cells against human tumor xenografts.

A variety of immunological approaches to cancer treatment are currently being explored. These include strategies designed to enhance or redirect the activity of T cells against tumors. Bispecific antibodies comprise a class of agents capable of redirecting T cells by binding to a tumor antigen and the T-cell receptor (TCR). In vivo pre-clinical testing of bispecific antibodies against human tumors has to date been limited to the use of immunodeficient mice that receive the bispecific agent, activated human effector T cells, and human tumor cells. In this report, we show that TCR transgenic/RAG-1 knockout mice (TCR/RAG) serve as a unique model allowing endogenous T cells to be redirected against transplanted human tumors. The findings show that TCR/RAG mice (i) accepted transplants of human tumors, including the folate-receptor-positive tumor line KB; (ii) contained endogenous cytotoxic T lymphocytes that could be activated in vivo with an antigenic peptide recognized by the transgenic TCR; (iii) rejected human tumors after treatment with the activating peptide and bispecific agents that contained folic acid co-valently linked to an anti-TCR antibody. Successful rejection was achieved with folate conjugates of Fab or scFv fragments. Treatment with activating agents and bispecific conjugates resulted in the complete eradication of freshly transplanted tumors as well as significantly prolonging the survival of mice bearing established solid tumors. Our results highlight the importance of including T-cell-activating modalities in combination with bispecific antibodies. Additionally, we introduce a system that allows endogenous T cells to be redirected against human tumor xenografts and in which the T cells may be followed in vivo by use of a clonotypic marker.

Intracerebral bispecific ligand-antibody conjugate increases survival of animals bearing endogenously arising brain tumors.

Bispecific antibodies capable of simultaneously binding a tumor surface antigen and the T-cell receptor/CD3 complex are capable of inducing polyclonal immune effector cells to destroy targeted tumor cells. Bispecific antibody immunotherapies have shown some promise against tumors of hematopoietic origin such as lymphomas, but use of bispecific antibodies for the treatment of solid tumors has been less fully explored. To test the preclinical potential of bispecific antibody therapy against an endogenously arising solid brain tumor, we have utilized a novel variation of conventional bispecific antibodies, referred to as bispecific ligand-antibody conjugates, to target choroid plexus tumors. The bispecific ligand-antibody conjugate described in this study is a chemical conjugate between an anti-CD3 monoclonal antibody (MAb) and folic acid, the ligand for a high-affinity surface receptor expressed on the surface of choroid plexus tumors. SV11 mice transgenic for SV40 large T antigen and its promoter develop solid choroid plexus tumors in the brain. We demonstrate that choroid plexus tumor cells are susceptible in vitro to cytolysis mediated by cytotoxic T cells in the presence of the bispecific ligand-antibody conjugate in a folate-inhibitable manner. Adoptive immunotherapy studies demonstrate the potential benefits of the bispecific ligand-antibody conjugate in vivo. The bispecific conjugate is capable of retaining adoptively transferred T lymphocytes specifically within tumor tissue for periods of up to at least 1 week. Further, following intracerebro-ventricular injection of bispecific conjugate and splenocytes containing activated cytotoxic T cells, T cells were observed to penetrate to interior regions of the tumor. A single treatment of adoptively delivered activated effectors and bispecific conjugate into the brain ventricles was insufficient to produce significant increases in survival of SV11 mice, but repeated treatment through indwelling cannulas prolonged survival of animals treated with activated effectors and bispecific ligand-antibody conjugate compared to animals treated with activated effectors or saline alone. Our results demonstrate that the SV11 model may be useful for preclinical evaluation and optimization of bispecific ligand-antibody conjugate treatments of solid tumors.

Targeting tumor cells with bispecific antibodies and T cells.

It has been known for some time that mammalian immune systems are capable of eliminating large tumor burdens. Redirecting the immune response of a patient to an established tumor has now become the focus of various therapeutic strategies. In this report, two projects toward this goal are described. The first project involves the development of a transgenic mouse model for T cell directed therapeutics. These mice express specific T cell receptor alpha and beta transgenes on a background in which the recombinational-activating-gene-1 (RAG) has been knocked out. The mice express cytotoxic T cells but not either T helper cells or B cells. Despite these deficiencies, the animals are capable of eliminating tumors that express the appropriate peptide/major histocompatibility complex ligand that is recognized by the alphabeta transgenic T cell receptor. Human tumors grow as transplants in these mice, thereby allowing various agents that redirect the endogenous T cells against human tumors to be tested. The second project involves a description of such agents: bispecific antibodies that simultaneously bind to an immune effector cell and a tumor cell. The bispecific antibody described here consists of folate attached to anti-T cell receptor antibodies, or their fragments. A single-chain Fv coupled with folate can redirect the lysis of human tumor cells that bear the high affinity folate receptor. Preliminary in vivo data showed that the folate/antibody conjugates were also capable of mediating rejection of the human tumor. This transgenic mouse model should now allow the evaluation and optimization of bispecific agents that can redirect a patient's own T cell response.

Targeting T cells against brain tumors with a bispecific ligand-antibody conjugate.

High-affinity receptors expressed on the surface of some tumors can be exploited by chemically conjugating the ligand for the receptor and an antibody against immune effector cells, thus redirecting their cytolytic potential against the tumor. Ovarian carcinomas and some brain tumors express the high-affinity folate receptor (FR). In this report, a transgenic mouse model that generates endogenously arising choroid plexus tumors was used to show that folate/anti-T-cell receptor antibody conjugates can direct infiltration of T cells into solid brain tumor masses. An engineered single-chain Fv form of the anti-T-cell receptor antibody KJ16 was conjugated with folate, to produce a bispecific agent that was substantially smaller than most previously characterized bispecific antibodies. Folate conjugation to the antibody increased T-cell infiltration into the tumors by 10- to 20-fold, and significantly prolonged survival of the mice.

Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells.

Bispecific antibodies that bind to a tumor antigen and the T cell receptor (TCR) redirect cytotoxic T lymphocytes (CTL) to lyse tumor cells which have escaped normal immune recognition mechanisms. One well-characterized tumor antigen, the folate receptor (FR), is expressed on most ovarian carcinomas and some types of brain cancer. Recently, it was shown that conjugates of folate and anti-TCR antibodies are extremely potent bispecific agents that target tumor cells expressing the high-affinity folate receptor, but not normal cells expressing only the reduced folate carrier protein. In this paper, it is shown that the size of these conjugates can be reduced to the smallest bispecific agent yet described (30 kDa) by attaching folate to a single-chain antibody, scFv, of the anti-TCR antibody KJ16. The scFv/folate conjugates are as effective as IgG/folate conjugates in mediating lysis of FR4 tumor cells by CTL. The optimal folate density was in the range of 5-15 folate molecules per scFv or IgG molecule, which yielded half-maximal lysis values (EC50) of approximately 40 pM (1.2 ng/mL for scFv). Finally, the scFv/folate conjugates could efficiently target tumor cells even in the presence of free folic acid at concentrations that are normally found in serum. Compared to conventional bispecific antibodies, the small size of scFv/folate conjugates may prove advantageous in the ability to penetrate tumors and in reduced immunogenicity.

Long-term adrenalectomy can decrease or increase hippocampal dentate gyrus volumes.

Male and female Long-Evans adult rats were adrenalectomized and sacrificed 6 weeks later to determine whether dentate gyrus damage would differ in females and males. A subset of adrenalectomized rats of both sexes had significantly reduced dentate gyrus volumes compared to the same sex SHAM operated rats. The remainder of the male and female adrenalectomized rats which did not have clear dentate gyrus damage had significantly larger dentate gyrus volumes compared to the same sex SHAM rats. The dentate gyrus volumes of all adrenalectomized rats were significantly correlated with two indices of residual hormonal levels (Na+/K+ ratios and body weight gain 6 weeks after surgery), indicating that endogenous corticosterone levels may be a determining factor in the response of the dentate gyrus to adrenalectomy. These dentate gyrus volumetric changes could not be attributed to tissue shrinkage as there were no changes in CA3 volumes in any of the groups. These results suggest that long-term adrenalectomy can result in either increased or decreased dentate gyrus volumes and that the adrenal steroid levels of each individual adrenalectomized rat may be the factor determining the direction of the dentate gyrus volumetric response.

Folate receptors as potential therapeutic targets in choroid plexus tumors of SV40 transgenic mice.

A high affinity folate receptor is expressed in some human cancers, including choroid plexus tumors and ependymomas, and has been suggested as a target for therapeutics. In this report, the expression of folate receptors in an SV40 large T antigen transgenic mouse (SV11) was investigated. SV11 mice develop choroid plexus tumors, a property that may be related to the observation that SV40 has been isolated from human choroid plexus tumors and ependymomas. We report that SV11 choroid plexus tumors contain a high affinity folate receptor (KD of 1 nM), detectable by 125I-folate autoradiography and immunohistochemistry. Western blot analysis indicated an apparent molecular weight of 38 kDa. RT-PCR revealed the presence of transcripts for both alpha and beta isoforms of the folate receptor. Brain parenchyma has undetectable folate receptor, but normal choroid plexus has substantial levels (as does human choroid plexus). The folate receptors of the tumor are accessible from the bloodstream whereas those of the normal choroid plexus are not. Thus SV11 transgenic mice should be useful for evaluating therapeutic targeting of high affinity folate receptors, both for efficacy of specific agents and possible side effects.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

Dentate gyrus destruction and spatial learning impairment after corticosteroid removal in young and middle-aged rats.

We investigated the functional and behavioral implications of chronic corticosteroid removal in young and middle-aged rats. Prepubertal and 13-month-old rats were adrenalectomized (ADX) or sham operated (SHAM). The young ADX rats were divided further into three groups: ADX with no hormone replacement, ADX given corticosterone chronically, (chCORT), and ADX given corticosterone acutely at the time of Morris water maze testing (acCORT). All rats were run on the Morris water maze 12 weeks after surgery. They were then sacrificed and the brains were removed for histological analysis. The results showed that prolonged corticosteroid absence caused major damage to the dentate gyrus and learning impairment on the Morris water maze. The chCORT rats had little dentate gyrus cell loss and were as efficient as the controls in Morris water maze performance, whereas the acCORT rats had dentate gyrus cell loss and were impaired in the spatial acquisition task. Furthermore, exogenously administered corticosterone had an interactive effect on ADX rats. Water maze performance was improved in dentate gyrus damaged rats (acCORT) compared to ADX rats not given corticosterone, whereas ADX rats with very little dentate gyrus damage (chCORT) did not exhibit better water maze performance relative to controls. Middle-aged ADX rats lost cells only in the dorsal blade of the dentate gyrus but they did not show a learning impairment in the Morris water maze relative to the middle-aged controls. These results indicate that corticosteroids are trophic for the dentate gyrus, that mature granule cells are less affected by adrenalectomy, that corticosteroid absence is responsible for some water maze impairment in ADX rats, but that in addition to corticosteroid absence, a substantial amount of dentate gyrus damage is necessary to impair spatial learning.

Selective loss of hippocampal granule cells following adrenalectomy: implications for spatial memory.

We examined the effects of long-term adrenalectomy (ADX) on hippocampal anatomy and behavioral learning in two spatial memory tasks. We assessed damage throughout the hippocampus by stereological analysis of the dentate gyrus and Ammon's horn. Rats were ADX or sham operated, and then tested in the Morris water maze 12 weeks after surgery, followed by testing on an eight-arm, alternating-baited radial maze at 22 weeks postsurgery. Animals were killed 7 1/2 months after surgery. ADX rats had selective volume reduction in the dentate gyrus with no changes in pyramidal regions CA1, CA2, CA3, or CA4. Dentate gyrus damage in some cases occurred throughout the entire rostrocaudal extent of the hippocampus. Analysis of corticosterone serum levels, serum Na+/K+ ratios, and body weight gain suggested that individual differences in dentate gyrus damage appear to be due to incomplete adrenalectomies or remaining ectopic tissue. ADX rats were able to learn in both the Morris water maze and eight-arm radial maze, even when the dentate gyrus was severely damaged (80% volume reduction). However, in the Morris water maze, the ADX rats' learning rate was significantly slower compared to controls. There was no difference between ADX and controls during reversal in either task. These data indicate that damage to the dentate gyrus following long-term ADX is severe enough to cause learning impairment in selected learning tasks. Such damage is restricted to the dentate gyrus and can occur throughout the rostrocaudal regions of the hippocampus.

Juvenile desipramine reduces adult sensitivity to imipramine in two behavioral tests.

The behavioral effects of adult imipramine administration were examined in female rats treated with desipramine as juveniles (JDES), treated with saline as juveniles (JSAL), and untreated as juveniles (JUNT). In the forced swimming test, the juvenile groups displayed similar behavioral effects of imipramine when administered short term following a pretest forced swimming exposure. Similar effects of imipramine were observed when administered long term prior to the only test exposure. When rats were not given a pretest forced swimming test exposure, short-term imipramine had no effect on JDES rats but did influence JSAL and JUNT rats. In the open-field test, short- and long-term imipramine treatment affected the behavior of JUNT and JSAL rats. Short-term imipramine treatment influenced open-field behavior of JDES animals, but long-term imipramine treatment had no effect. These results suggest that JDES treatment may permanently alter the neural mechanism underlying the behavioral effects of antidepressant treatment.

Progesterone-dependent sexual behavior and protein patterns in the ventromedial hypothalamus of the adult female rat.

Controversy exists concerning mechanisms by which progesterone exerts central nervous system effects on behavior. Progesterone may affect behavior by genomic regulation of protein synthesis. Alternatively, it may work through nongenomic mechanisms, consistent with its short latency to act. In the present study, we have examined the hypothesis that progesterone facilitation of sexual behavior is correlated with modification of the synthesis of specific proteins in the ventromedial hypothalamus (VMH). Ovariectomized rats were treated with either estradiol (4 mug/kg at 0 and 18 h) or estradiol (at 0 and 18 h) plus progesterone (2 mg/kg at 37 h). (35)S-labeled cysteine and methionine were bilaterally infused into the VMH at 37 h (the time of progesterone administration). Following 4 h of infusion, animals were tested for sexual behavior and sacrificed. Newly synthesized VMH proteins were separated by two-dimensional gel electrophoresis followed by fluorography. Analysis of approximately 660 spots/fluorogram in two independent replications indicated that no qualitative or quantitative changes in protein synthesis occurred in response to progesterone. In each replication statistical analysis suggested that the abundance of several proteins may have changed, but no specific proteins were changed in abundance in both replications. Within the range of this technique (10-100 kDa and 4.8-6.7 apparent pI) we found no evidence that progesterone causes alterations in VMH protein synthesis.

Characterization of a protein that appears in the nervous system of the moth Manduca sexta coincident with neuronal death.

Two-dimensional gel electrophoresis was used to locate potential neuronal death-related proteins in the moth Manduca sexta. Protein patterns of ganglia of pharate adult moths (taken prior to adult ecdysis) compared with protein patterns of one-day-old adults revealed reproducible changes in protein patterns. An acidic protein of approximately 40,000 Da was present in all samples from adult moths undergoing neuronal death and essentially absent from pharate adult samples.

Modulation by norepinephrine of neural responses to estradiol.

Pharmacological studies have suggested that neurotransmitter activity impinging on steroid-concentrating cells can affect the steroid receptor system within those cells, modifying behavioral responses to the hormone. The present experiments revealed that the alpha 1-noradrenergic antagonist prazosin, administered to ovariectomized rats at the time of each of two pulses of estradiol, inhibited the appearance of sexual receptivity. Prazosin also substantially reduced the levels of estrogen receptors within hypothalamic cell nuclei following an injection of estradiol. Manipulation of noradrenergic inputs into the hypothalamus by lesioning brain stem norepinephrine cell groups with 6-hydroxydopamine (6OHDA) also reduced the level of nuclear estrogen receptors following an injection of estradiol. Although this effect of 6OHDA lesions was observed in two separate experiments, in other experiments 6OHDA had no effect on estrogen receptors. In some instances, there was a positive correlation between nuclear estrogen receptor levels in the hypothalamus and the levels of norepinephrine. The results are consistent with the hypothesis that brain stem inputs to the hypothalamus are able to modulate neural responses to steroids and specifically that noradrenergic inputs are able to modulate neural responses to estradiol. However, there are additional undiscovered variables that preclude statements of a simple relationship between norepinephrine levels and estrogen receptor levels.