PCR to detect Mycobacterium tuberculosis in respiratory tract samples: evaluation of clinical data.

Tuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission in patients. The aims of this study were to evaluate two in-house molecular tests: nested PCR (nPCR) and real-time PCR (rtPCR) to detect M. tuberculosis complex directly from clinical samples. The results were compared to the culture results and to the culture results plus clinical data of patients. The rtPCR and nPCR presented high sensitivity (Se) and specificity (Sp) (rtPCR 97·6% and 91·5%, nPCR 85·7% and 92·7%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the Se and Sp were 90·2% and 97·3% for rtPCR and 80·4% and 98·6% for the nPCR, respectively. The results demonstrated that molecular assays of M. tuberculosis can provide a sensitive and rapid diagnostic of TB, and when used in addition to the clinical data of TB patients will help to improve the Sp of the diagnosis of pulmonary TB.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ¡Ý36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women.

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ≥36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

Interaction between midazolam-induced anterograde amnesia and memory enhancement by treatments given immediately after training on an inhibitory avoidance task in rats.

The aim of this study was to evaluate the effects of interactions between memory modulatory systems on inhibitory avoidance retention in rats. Adult female Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.3 mA footshock). The training-test interval was 24 h. The animals received an intraperitoneal injection of saline or midazolam (1 mg/kg) 15 min before training, and saline, adrenaline (25 microg/kg), naloxone (0.4 mg/kg), dexamethasone (0.3 mg/kg) or glucose (320 mg/kg) immediately after training. In saline-pretreated rats, adrenaline, naloxone, dexamethasone and glucose enhanced memory retention. Pretreatment with midazolam prevented the facilitatory effects of those treatments. These findings suggest that the facilitation of learning by post-training memory-enhancing treatments is prevented by midazolam.

Chronic treatment with clozapine, but not haloperidol, increases striatal ecto-5'-nucleotidase activity in rats.

In the search for differential mechanisms underlying clozapine's superior antipsychotic efficacy, the purinergic system has been considered, since an antagonist of the adenosine receptor A(2A) was shown to block clozapine acute effects on c-fos expression in rat striatum. Further investigating the interaction of clozapine with the purinergic system, we studied the effects of chronic treatment (28 days, intraperitoneal) with clozapine (25 mg/kg) and haloperidol (1.5 mg/kg) on the activity of ectonucleotidases in the striatum and hippocampus of rats. Clozapine selectively increased striatal 5'-nucleotidase activity (22%) compared to control and haloperidol groups. In vitro, neither drug affected enzyme activities. These results reinforce the differential effects of clozapine compared to haloperidol on the purinergic system.

Comparative pharmacological study of hydroethanol extracts of Passiflora alata and Passiflora edulis leaves.

Several species of the genus Passiflora, known in Brazil as "maracujá", have widespread use in folk medicine as sedatives and tranquillizers. The anxiolytic activity of hydroethanol extracts of P. alata and P. edulis leaves was evaluated using the elevated plus-maze test. The extracts presented anxiolytic activity in dosages around 50, 100 and 150 mg/kg.

Psychopharmacological screening of Pfaffia glomerata Spreng. (Amarathanceae) in rodents.

The alcoholic extract of Pfaffia glomerata roots (100, 500, 1000 mg/kg, intraperitoneally (i.p.), and 500, 1000, 1500 mg/kg, per os) was studied in several behavioral animal models for the evaluation of central activity: open field, barbiturate sleeping time, pentilenotetrazole (PTZ)-induced convulsions, elevated plus-maze, step-down inhibitory avoidance and forced swimming test. The acute treatment (500 mg/kg, i.p.) interfered with the open-field habituation, decreased sleep latency and increased barbiturate-induced sleeping time, protected partially the animals of PTZ-induced convulsions, decreased the memory retention in step-down inhibitory avoidance, and did not have an important effect in the elevated plus-maze test and forced swimming test. The same extract at 1000 mg/kg per os did not cause any effect in barbiturate sleeping time and pentilenotetrazole-induced convulsions models. Thus, the effect on the memory was deeper evaluated in the step-down inhibitory avoidance task. When administered by intraperitoneal route, the extract showed a dose-dependent effect causing full amnesia at 1000 mg/kg. On the other hand, when it was given by oral route at 500, 1000 and 1500 mg/kg, no influence on the memory retention was observed. These results suggest that the alcoholic extract of P. glomerata roots presents different effects depending on the route of administration: by i.p route, it seems to be a central nervous system depressant agent; by oral route, it seems to be ineffective, at least in the tested doses.

Role of hippocampal signaling pathways in long-term memory formation of a nonassociative learning task in the rat.

Long-term habituation to a novel environment is one of the most elementary forms of nonassociative learning. Here we studied the effect of pre- or posttraining intrahippocampal administration of drugs acting on specific molecular targets on the retention of habituation to a 5-min exposure to an open field measured 24 h later. We also determined whether the exposure to a novel environment resulted in the activation of the same intracellular signaling cascades previously shown to be activated during hippocampal-dependent associative learning. The immediate posttraining bilateral infusion of CNQX (1 microg/side), an AMPA/kainate glutamate receptor antagonist, or of muscimol (0.03 microg/side), a GABA(A) receptor agonist, into the CA1 region of the dorsal hippocampus impaired long-term memory of habituation. The NMDA receptor antagonist AP5 (5 microg/side) impaired habituation when infused 15 min before, but not when infused immediately after, the 5-min training session. In addition, KN-62 (3.6 ng/side), an inhibitor of calcium calmodulin-dependent protein kinase II (CaMKII), was amnesic when infused 15 min before or immediately and 3 h after training. In contrast, the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, and the protein synthesis inhibitor anisomycin, at doses that fully block memory formation of inhibitory avoidance learning, did not affect habituation to a novel environment. The detection of spatial novelty is associated with a sequential activation of PKA, ERKs (p44 and p42 MAPKs) and CaMKII and the phosphorylation of c-AMP responsive element-binding protein (CREB) in the hippocampus. These findings suggest that memory formation of spatial habituation depends on the functional integrity of NMDA and AMPA/kainate receptors and CaMKII activity in the CA1 region of the hippocampus and that the detection of spatial novelty is accompanied by the activation of at least three different hippocampal protein kinase signaling cascades.

Infusions of AP5 into the basolateral amygdala impair the formation, but not the expression, of step-down inhibitory avoidance.

We evaluated the effects of infusions of the NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) on the formation and expression of memory for inhibitory avoidance. Adult male Wistar rats (215-300 g) were implanted under thionembutal anesthesia (30 mg/kg, ip) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Bilateral infusions of AP5 (5.0 microg) were given 10 min prior to training, immediately after training, or 10 min prior to testing in a step-down inhibitory avoidance task (0.3 mA footshock, 24-h interval between training and the retention test session). Both pre- and post-training infusions of AP5 blocked retention test performance. When given prior to the test, AP5 did not affect retention. AP5 did not affect training performance, and a control experiment showed that the impairing effects were not due to alterations in footshock sensitivity. The results suggest that NMDA receptor activation in the BLA is involved in the formation, but not the expression, of memory for inhibitory avoidance in rats. However, the results do not necessarily imply that the role of NMDA receptors in the BLA is to mediate long-term storage of fear-motivated memory within the amygdala.

The anticonvulsant compound gabapentin possesses anxiolytic but not amnesic effects in rats.

This report describes the effects of the antiepileptic agent gabapentin on anxiety and memory. Male Wistar rats received intraperitoneal administrations of gabapentin (10, 30 and 100mg/kg), diazepam (1 mg/kg), saline or diazepam vehicle 30 minutes prior to experimental procedures. Animals were: (1) tested on step-down inhibitory avoidance (footshock 0.3 mA) and habituation to an open-field for memory assessment; and (2) submitted to the elevated plus-maze to evaluate the potential anxiolytic effects of gabapentin. Animals treated with gabapentin showed a reduction in anxiety similar to that observed in animals treated with diazepam. Memory was not affected by gabapentin in any of the tests, but was impaired by diazepam. The lack of effects of gabapentin on memory suggest a potential advantage of this drug over compounds with previously known anxiolytic property, which have amnesic effects at doses used for the treatment of anxiety disorders.

NMDA receptor antagonism in the basolateral amygdala blocks enhancement of inhibitory avoidance learning in previously trained rats.

Extensive evidence suggests that N-methyl-D-aspartate (NMDA) glutamate receptor channels in the amygdala are involved in fear-motivated learning, and infusion of NMDA receptor antagonists into the amygdala blocks memory of fear-motivated tasks. Recent studies have shown that previous training can prevent the amnestic effects of NMDA receptor antagonists on spatial learning. In the present study, we evaluated whether infusion of the NMDA antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) impairs reinforcement of inhibitory avoidance learning in rats given previous training. Adult male Wistar rats (220-310 g) were bilaterally implanted under thionembutal anesthesia (30 mg/kg, i.p.) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Infusion of AP5 (5.0 microg) 10 min prior to training in a step-down inhibitory avoidance task (0.4 mA footshock) blocked retention measured 24 h after training. When infused 10 min prior to a second training session in animals given previous training (0.2 mA footshock), AP5 blocked the enhancement of retention induced by the second training. Control experiments showed that the effects were not due to alterations in motor activity or footshock sensitivity. The results suggest that NMDA receptors in the basolateral amygdala are involved in both formation of memory for inhibitory avoidance and enhancement of retention in rats given previous training.

Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats.

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation.

Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29 percent increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation

Normal inhibitory avoidance learning and anxiety, but increased locomotor activity in mice devoid of PrP(C).

Prions are the causative agents of transmissible spongiform encephalopathies. The transmissible agent (PrP(Sc)) is an abnormal form of PrP(C), a normal neuronal protein. The physiological role of PrP(C) remains unclear. In the present report, we evaluated behavioral parameters in Prnp(0/0) mice devoid of PrP(C). Prnp(0/0) mice showed normal short- and long-term retention of a step-down inhibitory avoidance task and normal behavior in an elevated plus maze test of anxiety. During a 5-min exploration of an open field, Prnp(0/0) mice showed normal number of rearings, defecation, and latency to initiate locomotion, but a significant increase in the number of crossings. The results suggest that Prnp(0/0) mice show normal fear-motivated memory, anxiety and exploratory behavior, and a slight increase in locomotor activity during exploration of a novel environment.

Memory-enhancing treatments do not reverse the impairment of inhibitory avoidance retention induced by NMDA receptor blockade.

The aim of the present research was to verify whether the impairment of retention induced by the N-methyl-d-aspartate (NMDA) receptor blocker (+)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]cycloheptene-5,10 imine (MK-801) can be reversed by memory-enhancing treatments. Adult female Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.3-mA foot shock, 24-h training-test interval). Animals were given an ip injection of saline (SAL) or MK-801 (0.0625 mg/kg) 30 minutes before training, and an ip injection of SAL, epinephrine (EPI) (25 microg/kg), the opioid receptor antagonist naloxone (NAL) (0.4 mg/kg), the glucocorticoid receptor agonist dexamethasone (DEX) (0.3 mg/kg), or glucose (GLU) (320 mg/kg) immediately after training. There was an impairment of inhibitory avoidance retention in the MK-801-SAL, MK-801-EPI, MK-801-NAL, MK-801-DEX, and MK-801-GLU groups. There was an enhancement of retention in the SAL-EPI, SAL-NAL, SAL-DEX, and SAL-GLU groups. A control experiment showed that the amnestic effects of MK-801 could not be attributed to decreased reactivity to the foot shock. The results suggest that memory-enhancing treatments directed at modulatory mechanisms do not reverse the memory impairment induced by NMDA receptor blockade.

Differential effects of post-training muscimol and AP5 infusions into different regions of the cingulate cortex on retention for inhibitory avoidance in rats.

Adult male Wistar rats were bilaterally implanted with indwelling cannulae in four different coordinates of the cingulate cortex: (1) the anterior cingulate (AC), (2) the rostral region of the posterior cingulate (RC), (3) the upper portion of the caudal region of the posterior cingulate (UC), and (4) the lower portion of the caudal region of the posterior cingulate (LC). After recovery, animals were trained in a step-down inhibitory avoidance task (3.0-s, 0.4-mA foot shock). Either immediately, or 90 or 180 min after training, animals received a 0.5-microl infusion of vehicle (phosphate buffer, pH 7.4), of muscimol (0.5 microg), or of AP5 (5.0 microg). Retention testing was carried out 24 h after training. Muscimol was amnestic when given into any of the three coordinates of the posterior cingulate cortex 90 min after training, and when given into LC immediately post-training. In addition, AP5 was amnestic when given into UC 90 min post-training, but not when given into any other region and/or at any other time. None of the treatments had any effect when given into AC. The results suggest that memory processing of the inhibitory avoidance task is regulated by the posterior but not by the anterior cingulate cortex, through muscimol-sensitive synapses, relatively late after training. AP5-sensitive synapses appear to play a very limited role in these processes, restricted to UC.

Two time windows of anisomycin-induced amnesia for inhibitory avoidance training in rats: protection from amnesia by pretraining but not pre-exposure to the task apparatus.

We have studied the effect of training conditions on hippocampal protein synthesis-dependent processes in consolidation of the inhibitory avoidance task. Adult male Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.4 mA foot shock, 24 hr training-test interval). Fifteen minutes before or 0, 3, or 6 hr after training, animals received a 0.8-microl intrahippocampal infusion of the protein-synthesis inhibitor anisomycin (80 microg) or vehicle (PBS, pH 7.4). The infusion of anisomycin impaired retention test performance in animals injected 15 min before and 3 hr after the training session, but not at 0 or 6 h post-training. Pretraining with a low foot shock intensity (0.2 mA) 24 hr before training, prevented the amnestic effect of anisomycin injected at 15 min before or 3 hr after training. However, simple pre-exposure to the inhibitory avoidance apparatus did not alter the amestic effects of anisomycin. The results suggest that hippocampal protein synthesis is critical in two periods, around the time of, and 3 hr after training. A prior weak training session, however, which does not itself alter step-down latencies, is sufficient to prevent the amnestic effect of anisomycin, suggesting that even if not behaviorally detectable, weak training must be sufficient to produce some lasting cellular expression of the experience.