Using FT-IR spectroscopy for the identification of the T. cruzi, T. rangeli, and the L. chagasi species.

The cross-reaction in the diagnosis results is a serious problem, leading to an incorrect treatment and several injuries to patients. The Trypanosoma rangeli and Trypanosoma cruzi belong to the genus Trypanosoma, but the Trypanosoma rangeli is a non-pathogenic parasite to humans. While Trypanosoma cruzi is the etiological agent of Chagas' disease, which affects circa 2-3 million people and more than 6000 deaths annually in Brazil. The Leishmania chagasi causes infectious disease known as visceral leishmaniasis. This diseases have in common the crossed antigenic reaction promoted by serological tests and its differentiation is relevant for epidemiological studies and clinical practice. In this study the Fourier Transform Infrared (FT-IR) Spectroscopy was used to differentiate these microorganisms, which were cultivated and the spectra analyzed. Data analysis were performed by Gaussian curve fitting and multivariate statistical analysis. The cluster analysis have shown four specific regions to identify the microorganisms. The first three PCs of principal component analysis associated to linear discriminant were able to classify 95.6% of the parasites using cross-validation. The curve fitting method showed the quantitative differentiation among L. chagasi, T. cruzi, and T. rangeli species in the vibrational regions of polysaccharides, amide III, lipid esters, and fatty acid.

Fluoxetine reduces periodontal disease progression in a conditioned fear stress model in rats.

BACKGROUND AND OBJECTIVE: Recent evidence suggests that the use of fluoxetine could reduce periodontal disease severity. However, the effect of fluoxetine on periodontal disease has not been tested in the context of conditioned fear stress (CFS). We hypothesized that inhibition of chronic stress by fluoxetine might decrease the levels of bone loss in periodontal disease. The aim of the present study was to analyze the effect of fluoxetine on bone loss in chronic periodontitis. MATERIAL AND METHODS: Fourteen Wistar rats were submitted to ligature-induced periodontal disease and divided into four groups (A-D). Groups A (n = 3) and B (n = 4) were not stressed, while Groups C (n = 3) and D (n = 4) were submitted to a CFS paradigm for 38 d. Daily fluoxetine (20 mg/kg) was administered to Groups B and D from day 20 to day 39, at which point the rats were submitted to an open field test and killed on day 40. Mandibles were removed for histological and immunohistochemical analyses. RESULTS: Stress was associated with a higher level of bone loss in Group C compared with Group A. Additionally, no differences in bone loss were observed among Groups A, B and D. CONCLUSION: We showed that stress is associated with the progression of bone loss in a CFS model in rats and that fluoxetine treatment reduces the bone loss.

Diazepam reverses the alveolar bone loss and hippocampal interleukin-1beta and interleukin-6 enhanced by conditioned fear stress in ligature-induced periodontal disease in rats.

BACKGROUND AND OBJECTIVE: Stress and anxiety have been associated with chronic periodontitis, but few studies examining the effects of psychotropic drugs on periodontal health have been performed. Therefore, we aimed to investigate the effects of diazepam on the progression of periodontitis in chronically stressed rats. MATERIAL AND METHODS: Fourteen Wistar rats were submitted to ligature-induced periodontal disease and were divided into four groups . Two groups were not stressed, whereas two groups were submitted to a conditioned fear stress paradigm for 38 d. Daily diazepam treatment (2 mg/kg, orally) was administered to one unstressed group and to one group submitted to a conditioned fear stress paradigm from day 2 to the day 39, at which point the rats were submitted to an open field test and were killed on day 40. Brains and mandibles were removed for histological and immunohistochemical analyses. RESULTS: Animals exposed to conditioned fear stress presented an increase in freezing behavior, a decrease in locomotor activity, enhanced alveolar bone loss and higher levels of hippocampal interleukin-1beta (IL-1ß) and interleukin-6 (IL-6), compared with the control group. Diazepam, at the dose used in the current study, had no effect on freezing behavior but reversed the decrease in locomotor activity provoked by stress. Additionally, the treatment reduced the levels of hippocampal IL-1ß and IL-6 and alveolar bone loss in Wistar rats. Neither conditioned fear stress nor diazepam treatment had an effect on periodontal IL-1ß or IL-6 levels in animals. CONCLUSION: Our results suggest that diazepam treatment reduces bone loss in rats submitted to conditioned fear stress. In addition, diazepam treatment led to decreased IL-1ß and IL-6 levels in the hippocampus.

The characterization of two monoclonal antibodies which react with high molecular weight antigens of asexual Plasmodium falciparum.

We prepared rat monoclonal antibodies (mAb) specific for very large Plasmodium falciparum proteins to assist in their characterization. Hybridomas prepared from rats immunized with parasitized erythrocyte (PE) proteins of greater than 200 kDa exhibited two patterns of Western blot reactivity with PE SDS extracts: one represented by clone 41E11 (IgM, kappa), the other by clone 12C11 (IgM, lambda). MAb 41E11 reacted by Western blotting with at least 15 antigens, most of which comigrated with antigens identified by the 33G2 human IgM mAb. The stage specificity of mAb 41E11 reactivity and indirect immunofluorescence (IFA) pattern closely resemble those previously described for antigens that share the EEXXEE sequence motif. Unlike mAb 33G2, MAb 41E11 immunoprecipitated a biosynthetically radiolabeled protein of 320 kDa. MAb 41E11 did not immunoprecipitate any cell surface 125I proteins. MAb 12C11 reacted on Western blotting with a different group of malarial antigens of approximately 44, 95, 117, 145, and 310 kDa, as well as with some low-molecular-weight, uninfected erythrocyte antigens. MAb 12C11 did not immunoprecipitate any cell surface 125I or biosynthetically labeled proteins. The 310-kDa antigen recognized by mAb 12C11 (denoted Ag 12A) does not correspond to PfEMP2 or the 320-kDa antigen recognized by mAbs 33G2 or 41E11. With trophozoites and more mature stages, fixed IFA reactivity of mAb 12C11 was at the parasite and in antigen aggregates in the host cell cytoplasm that extended to the PE plasma membrane. Indirect results suggest that Ag 12A does not correspond to cell surface-exposed PfEMP1 and is most likely a hitherto unidentified malarial protein.

Role of 5-hydroxytryptamine in amphetamine effects on punished and unpunished behaviour.

In order to assess the contribution of serotonergic (5-HT) mechanisms in the suppressant effect of amphetamine on punished responding, dose-effect curves of amphetamine on key-pecking behaviour of pigeons maintained by food presentation and punished by electric-shock were determined before and after pretreatment with methergoline, a potent and specific 5-HT receptor blocker in the central nervous system. A multiple fixed-interval 5 min, fixed-interval 5 min schedule of reinforcement in which every response, except the reinforced one, was punished in one of the two components (mult FI5 FI5-shock) was used. Effective doses of amphetamine decreased unpunished as well as punished FI response rates. However, the decreases in punished behaviour were more evident and dose-dependent. Methergoline markedly increased FI responding in the punished FI component but only slightly increased or decreased unpunished FI response rates. The most effective dose of methergoline for increasing punished responding was 0.56 mg/kg. Pretreatment with this dose of methergoline unmasked the facilitatory effects of amphetamine on unpunished responding, but did not antagonize its suppressant effect on punished responding. Therefore, although 5-HT seems to mediate punishment-induced response suppression and to inhibit the facilitatory effects of amphetamine on unpunished responding, it is not apparently involved in the suppressant effect of amphetamine on punished behaviour.

Anti-aversive role of serotonin in the dorsal periaqueductal grey matter.

Microinjection of 5, 10, and 20 nmol serotonin (5-HT) and of 0.5, 1, and 2 nmol 5-methoxy-N, N-dimethyltryptamine (5-MeODMT) into the dorsal midbrain of rats bearing chronically implanted chemitrodes raised the electrical threshold for inducing escape behaviour following stimulation of the dorsal periaqueductal grey matter (DPAG). Linear regressions of log dose against drug-induced increase in aversive threshold were obtained for 5-HT and 5-MeODMT. The 5-MeODMT dose-effect curve was steeper and lay to the left of the 5-HT dose-effect curve. Local pre-treatment with 10 nmol metergoline or ketanserin blocked the anti-aversive effect of 10 nmol 5-HT, whereas pre-treatment with 100 nmol zimelidine potentiated this effect of 5-HT. The same dose of zimelidine raised the aversive threshold when given alone. These results suggest that 5-HT plays an inhibitory role in the DPAG controlling aversion, probably mediated by 5-HT2 receptors.

Role of benzodiazepine receptors located in the dorsal periaqueductal grey of rats in anxiety.

Studies with electrical brain stimulation suggest that the dorsal periaqueductal grey matter (DPAG) is related to anxiety and to the anti-aversive effects of benzodiazepines (BZD) compounds. However, direct stimulation of the brain may prevent conclusions about the role of specific regions in the control of normal behaviour. In the present study we employed the elevated plus-maze, an ethologically based model of anxiety, to investigate the role of BZD receptors located in the DPAG in anxiety and in the anxiolytic effect of systemically injected BZD. The results showed that midazolam (20-80 nmol), a BZD agonist, dose-dependently increased the percentage of entries and time spent in open arms when microinjected into the DPAG. The effect of midazolam (80 nmol) was antagonized by flumazenil (80 nmol), a BZD antagonist, microinjected into the DPAG 10 min before the agonist. FG 7142 (20-80 nmol), a BZD partial inverse agonist, decreased time spent in open arms at the dose of 40 nmol and the number of open arms entries at all doses when microinjected into the DPAG. The microinjection of flumazenil (80 nmol) into the DPAG failed to antagonize the anxiolytic effect of systemically injected diazepam (2.5 mg/kg). These results strengthen the idea of an involvement of BZD receptors located in the DPAG with anxiety. They also suggest that the DPAG is not the only structure responsible for the anxiolytic effects of systemically injected BZD.

Anxiolytic effect in the elevated plus-maze of the NMDA receptor antagonist AP7 microinjected into the dorsal periaqueductal grey.

In order to localise the often reported anxiolytic action of N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-7-phosphonoheptanoic acid (AP7) was injected into the dorsal periaqueductal grey (DPAG) of rats exposed to the elevated plus-maze model of anxiety. Doses of 0.2, 2 and 20 nmol AP7 caused a dose-dependent increase in the percentage of open arm entries, the effect of the last two doses being significantly different from control. A non-significant tendency to increase the percentage of time spent on the open arms of the maze was also noticed. In contrast, the total number of entries into either the open or enclosed arms was not affected. Injections of AP7 localized outside the DPAG were ineffective. Therefore, microinjection of AP7 into the DPAG caused a selective anxiolytic effect in the elevated plus-maze. It may be suggested that the DPAG is a site of the anxiolytic action of NMDA antagonists reported following systemic administration.

Anxiolytic effect of nitric oxide synthase inhibitors microinjected into the dorsal central grey.

Nitric oxide synthase (NOS) accounts for most of the NADPH-diaphorase neuronal activity in the brain. NADPH-diaphorase-positive neurones have been localized at the dorso-lateral part of the periaqueductal grey (PAG), a region related to anxiety. Microinjections of the NOS inhibitors L-NAME (10-200 nmol 0.5 microliter-1) and L-NOARG (10-100 nmol) at this site induced anxiolytic-like effects in the elevated plus maze. These effects, however, occurred only at a limited range of doses and the dose-effect curve had a bell shape, higher doses of both compounds tending to be anxiogenic. The anxiolytic effect of L-NAME was antagonized by a previous microinjection of L-arginine (50 nmol 0.5 microliter-1). These results suggest that NO may play a role in PAG neurotransmission involved in anxiety.

An improved microassay for Plasmodium falciparum cytoadherence using stable transformants of Chinese hamster ovary cells expressing CD36 or intercellular adhesion molecule-1.

Stable transformants of Chinese hamster ovary (CHO) cell lines expressing high levels of human CD36 or intercellular adhesion molecule-1 (ICAM-1) have been produced as target cells for cytoadherence of Plasmodium falciparum-infected erythrocytes. An improved adherence microassay has been designed using small sample volumes and allowing convenient and reliable measurements on a large number of samples. The assay can be used both with purified proteins spotted on plastic and with the stably transformed CHO cell lines. The same assay plate can be evaluated either microscopically or by scintillation counting after use of 3H-hypoxanthine-labeled parasites. Using the microassay, functional expression of the transfected receptor molecules on CHO-CD36 and CHO-ICAM was confirmed using parasites with different cytoadherence phenotypes and cytoadherence inhibition experiments with a panel of anti-CD36 antibodies. The use of isolates from The Gambia confirmed the applicability of these assays for laboratory studies of these isolates.

Surface molecules on Plasmodium falciparum-infected erythrocytes involved in adherence.

The identity of cell surface receptor molecules on Plasmodium falciparum-infected erythrocytes is of great interest since the functional sites involved in attachment to endothelial cells may be structurally conserved in wild isolates. Such conserved sites may represent suitable antigenic targets for a vaccine-induced immune response that would block or reverse infected cell sequestration in vivo. Identification of the infected cell receptor sites may also lead to novel methods for treatment of acute cerebral malaria. We review the likely roles, either direct or indirect, for the participation of knob protrusions, malarial proteins expressed at the cell surface, and modified host membrane proteins in the specific receptor properties acquired by infected erythrocytes.

Agglutination of Plasmodium falciparum-infected erythrocytes from east and west African isolates by human sera from distant geographic regions.

Plasmodium falciparum-infected erythrocytes (PfE) were collected from acutely infected children in The Gambia and Tanzania and cultured for more than 30 hr until the parasites were mature trophozoites. Sera collected from these countries, other African countries, Asia, and South America were used in the PfE microagglutination test to determine whether PfE from East and West Africa share surface antigens. From the patterns of agglutination reactivity, we identified extensive antigenic diversity in surface antigens, but obtained no evidence for greater differences between isolates from East or West Africa and those within one region. The majority of sera from immune adults from The Gambia, Tanzania, Sudan, Nigeria, or Ghana were pan-agglutinating, and agglutinated all PfE isolates from The Gambia and Tanzania. Some sera from immune adults of Irian Jaya also agglutinated each of the seven African isolates, while others agglutinated many but not all of the isolates, similar to sera from immune adults of Flores, Indonesia. In contrast, sera from nonimmune adults from Colombia agglutinated few of the African isolates. It was remarkable, however, that sera from nonimmune Colombians agglutinated any African isolates. Our results are consistent with the following conclusions: some PfE surface antigen(s) are very diverse; this diversity is a feature of the parasite worldwide; the repertoire of isolate-specific surface antigens, although large, includes antigens that are either identical or antigenically cross-reactive in geographically very distant parasite populations; and African adults have pan-agglutinating antibodies that may contribute to protective immunity. Such pan-agglutinating antibodies could reflect the accumulation of a large repertoire of isolate-specific antibodies. The contribution of antibody against any shared PfE surface antigen to the pan-agglutinating reactivities is unknown and awaits development of the appropriate reagents.

GABA modulation of the defense reaction induced by brain electrical stimulation.

Earlier behavioral results led to the suggestion that GABA exerts a tonic inhibitory influence in the dorsal periaqueductal gray (DPAG) matter of the rat integrating defensive behavior. In the present experiments, the role of GABAergic mechanisms in the modulation of the autonomic component of the defense reaction was studied. Thus, the effects of intravenous (IV) injections of chlordiazepoxide as well as of intracerebral (IC) injections of midazolam in the dorsal midbrain, on the blood pressure (BP), heart rate (HR) and respiratory increases induced by electrical stimulation of the DPAG were measured in rats anesthetized with urethane. Chlordiazepoxide (10 mg/kg, IV) as well as midazolam (40 and 160 nmol, IC) attenuated the centrally-induced hypertension, without affecting basal BP. The tachycardia induced by aversive brain stimulation was similarly decreased by the benzodiazepines. In addition, the HR baseline was significantly raised by chlordiazepoxide and by the highest dose of midazolam. The tachypnea induced by brain electrical stimulation was also reduced by both benzodiazepines. Basal respiratory rate was slightly, but significantly decreased by chlordiazepoxide as well as by the two doses of midazolam used and to a lesser extent by the vehicle alone. Chlordiazepoxide attenuated the increase in respiratory depth caused by brain stimulation, while basal respiratory amplitude was not affected. The effects of midazolam on this parameter were unclear. Microinjection of bicuculline (5 and 10 nmol) or picrotoxin (0.3 and 1 nmol) into the DPAG increased the BP, HR and respiration, like the electrical stimulation. The latency and duration of bucuculline effects were shorter than those of picrotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro expression and in vivo immunogenicity of Plasmodium falciparum pre-erythrocytic stage DNA vaccines.

DNA vaccine plasmids were constructed that encoded four pre-erythrocytic antigens from the human malaria parasite Plasmodium falciparum: circumsporozoite protein (PfCSP); sporozoite surface protein 2 (PfSSP2); carboxyl terminus of liver stage antigen 1 (PfLSA-1 c-term); and, exported protein 1 (PfExp-1). Antigen expression was evaluated in vitro by immunoblot analysis of tissue culture cells following transient transfection with each plasmid. Clearly detectable levels of expression depended upon, or were markedly enhanced by, fusion of the antigen encoding sequences in-frame with the initiation complex and peptide leader sequence of human tissue plasminogen activator protein. Mice injected with these plasmids produced antigen specific antibody and cytotoxic T lymphocyte responses. However, the magnitudes of the responses were not always predicted by the in vitro expression assay. The results of this study provided the basis for further testing of these plasmids in primates and the formulation of multi-component pre-erythrocytic DNA vaccines for efficacy testing in human volunteers.

Mediation by serotonin of the antiaversive effect of zimelidine and propranolol injected into the dorsal midbrain central grey.

Previously reported results indicate that serotonin (5-HT) inhibits the neural sub strate of aversion in the dorsal midbrain central grey (DCG) of the rat. In addition, the present results show that microinjection of the 5-HT uptake inhibitor zimelidine (100 nmol) into the DCG of rats with chronically implanted chemitrodes raised the threshold of aversive electrical stimulation. This antiaversive effect of zimelidine was antagonized by pretreatment with the 5-HT(2) receptor blocker ritanserin (10 nmol), also microinjected into the DCG. In contrast, the antiaversive effect of the benzodiazepine agonist midazolam (40 nmol) was unaffected by ritanserin. Propranolol (2.2, 4.4 and 8.8 nmol) raised the aversive threshold in a dose-depen dent way following its injection into the DCG. The antiaversive effect of 4.4 nmol of propranolol was antagonized by previous administration of ritanserin (10 nmol). Moreover, combined administration of zimelidine (100 nmol) followed by propranolol (4.4 nmol) caused an anti aversive effect which was equivalent to the sum of the effect of each drug alone. These results indicate that the antiaversive effect of intracerebrally injected zimelidine and propranolol is mediated by endogenous 5-HT, through activation of 5-HT(2) receptors.

DNA vaccines against malaria: immunogenicity and protection in a rodent model.

Since the first demonstration of the technology a few years ago, DNA vaccines have emerged as a promising method of vaccination. In a variety of experimental systems, DNA vaccines have been shown not only to induce potent immune responses, but also to offer many advantages in terms of ease of construction, testing, and production. In this article we summarize the progress achieved in development of DNA vaccines that can protect mice from infection by the rodent malaria parasite Plasmodium yoelii, describe initial studies of immunogenicity of a malaria DNA vaccine in a primate model, and outline the strategies being employed to design the next generation of malaria DNA vaccines.

Effect of minor tranquilizers, tryptamine antagonists and amphetamine on behavior punished by brain stimulation.

Earlier observations have shown that septal lesions released operant responding punished by foot-shock, but did not change behavior punished by electrical stimulation of the dorsal periaqueductal gray (DPAG) substance of the rat brain. In contrast, chlordiazepoxide facilitated both kinds of punished responding. In order to further study the mechanism of brain stimulation punishment, dose-response curves of two minor tranquilizers, chlordiazepoxide and pentobarbital, of two tryptamine antagonists, methysergide and cyproheptadine as well as of amphetamine on lever-pressing behavior of rats maintained by water reinforcement and punished by DPAG stimulation were determined. A multiple schedule with a variable-interval 2 min (VI 2) non-punished component and a continuous reinforcement (CRF) component in which every response was both rewarded and punished was used. Chlordiazepoxide and pentobarbital caused dose-dependent increases in punished responding. Unpunished VI response rates were also moderately increased by the minor tranquilizers. In contrast, neither methysergide nor cyproheptadine increased punished or unpunished responding at doses that have been previously shown to markedly release behavior punished by foot-shock, in the rat. Conversely, amphetamine, a drug that usually does not release responding punished by peripheral noxious stimulation, caused dose-dependent increases in responding suppressed by DPAG punishment without affecting VI response rate. These and previous results with septal lesions suggest that neither the septo-hippocampal system nor its serotonergic input from the mesencephalon mediate response suppression by DPAG electrical stimulation, in contrast to their active role in peripheral punishment. This difference may also explain the marked facilitatory effect of amphetamine on responding punished by brain stimulation shown by the present results.

GABA mediation of the anti-aversive action of minor tranquilizers.

Earlier observations have shown that systematically injected minor tranquilizers decrease the aversive consequences of electrical stimulation of the dorsal periaqueductal gray (DPAG) matter of the rat brain. In order to verify if these drugs can act directly on the DPAG, chlordiazepoxide (CDP) and pentobarbital (PB) were locally injected into the dorsal midbrain of rats chronically implanted with chemitrodes, allowing electrical stimulation of the same brain area. Microinjection of doses of 0.16 and 0.32 mumol of CDP and 0.16 mumol of PB significantly increased the threshold electrical current including flight behavior by stimulating the dorsal midbrain. Flight behavior was measured by the number of times rats crossed dividing line while running from one compartment of a shuttle-box to the other. The same effect was caused by the intracerebral injection of 0.32 and 0.64 mumol of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). Conversely, local injection of the GABA antagonists. bicuculline (5-20) nmol) or picrotoxin (0.3 and 0.6 nmol), into the dorsal midbrain induced flight behavior, like the electrical stimulation. On the other hand, the glycine antagonist, strychnine (40 nmol) caused convulsive behavior only, while the intracerebral injection of the cholinergic agonist, carbachol (10-40 nmol), increased locomotion, sniffing and turning behavior, but did not induce flight. Pretreatment with locally injected GABA (0.64 mumol) antagonized the aversive effect of either bicuculline (10 nmol) or picrotoxin (0.3 nmol), whereas CDP (0.32 mumol) antagonised bicuculline only and PB (0.16 mumol) was ineffective against either bicuculline or picrotoxin. These results suggest that minor tranquilizers act directly upon the DPAG by enhancing the tonic inhibitory influence of endogenous GABA. This action may underly the antiaversive affects of these drugs.

Depressant action of chlordiazepoxide on cardiovascular and respiratory changes induced by aversive electrical stimulation of the brain.

1. Electrical stimulation of the dorsal periaqueductal gray matter (DPAG), an aversive area of the rat brain, increased the mean blood pressure of awake rats as well as of animals anesthetized with urethane. 2. In the anesthetized rats, increases in heart rate and in breath rate were also induced by DPAG stimulation. 3. Chlordiazepoxide, a benzodiazepine, decreased the blood pressure rise caused by aversive stimulation of the brain in the awake rat. 4. Chlordiazepoxide elicited the same effect in urethane-anesthetized rats. In addition, the hyperpnea induced by electrical stimulation of the dorsal periaqueductal gray matter was also decreased by the drug. 5. The pressor response to intravenous noradrenaline was not affected by chlordiazepoxide. 6. These results suggest that benzodiazepines attenuate the neurovegetative changes accompanying emotion by depressing brain systems that integrate emotional behavior.

Anxiolytic effect of midazolam microinjected into the dorsal periaqueductal grey area of rats.

In order to investigate the role of the dorsal periaqueductal grey (DPAG) area in the anxiolytic effect of benzodiazepines male Wistar rats (N = 10), weighing 200-250 g at the time of surgery, were microinjected into this structure with midazolam (80 nmol) and submitted to the elevated plus-maze, an ethologically based model of anxiety. Midazolam significantly increased the percentage of open arm entries from 32.4 +/- 4.6 (control) to 49.5 +/- 3.0 and of time spent in the open arms from 21.0 +/- 4.5 (control) to 35.6 +/- 4.8 without affecting the total number of entries into either open or enclosed arms. This effect typifies an anxiolytic effect in the test and was antagonized by the benzodiazepine receptor antagonist flumazenil (80 nmol) microinjected into the same site 10 min before the midazolam (80 nmol) microinjection. Microinjection of flumazenil alone had no effect. These results provide additional evidence for the participation of the DPAG in the physiopathology of anxiety and suggest that it may be a site for the anxiolytic effect of systemically injected benzodiazepines.