McFarland nephelometer as a simple method to estimate the sensitivity of the polymerase chain reaction using Mycobacterium tuberculosis as a research tool

Polymerase chain reaction (PCR) has been widely investigated for the diagnosis of tuberculosis. However, before this technique is applied on clinical samples, it needs to be well standardized. We describe the use of McFarland nephelometer, a very simple approach to determine microorganism concentration in solution, for PCR standardization and DNA quantitation, using Mycobacterium tuberculosis as a model. Tuberculosis is an extremely important disease for the public health system in developing countries and, with the advent of AIDS, it has also become an important public health problem in developed countries. Using Mycobacterium tuberculosis as a research model, we were able to detect 3 M. tuberculosis genomes using the McFarland nephelometer to assess micobacterial concentration. We have shown here that McFarland nephelometer is an easy and reliable procedure to determine PCR sensitivity at lower costs.

The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze

The present study evaluated the correlation between the behavior of mice in the forced swimming test (FST) and in the elevated plus-maze (PM). The effect of the order of the experiments, i.e., the influence of the first test (FST or PM) on mouse behavior in the second test (PM or FST, respectively) was compared to handled animals (HAND). The execution of FST one week before the plus-maze (FST-PM, N = 10), in comparison to mice that were only handled (HAND-PM, N = 10) in week 1, decreased % open entries (HAND-PM: 33.6 ± 2.9; FST-PM: 20.0 ± 3.9; mean ± SEM; P<0.02) and % open time (HAND-PM: 18.9 ± 3.3; FST-PM: 9.0 ± 1.9; P<0.03), suggesting an anxiogenic effect. No significant effect was seen in the number of closed arm entries (FST-PM: 9.5 (7.0-11.0); HAND-PM: 10.0 (4.0-14.5), median (interquartile range); U = 46.5; P>0.10). A prior test in the plus-maze (PM-FST) did not change % immobility time in the FST when compared to the HAND-FST group (HAND-FST: 57.7 ± 3.9; PM-FST: 65.7 ± 3.2; mean ± SEM; P>0.10). Since these data suggest that there is an order effect, the correlation was evaluated separately with each test sequence: FST-PM (N = 20) and PM-FST (N = 18). There was no significant correlation between % immobility time in the FST and plus-maze indexes (% time and entries in open arms) in any test sequence (r: -0.07 to 0.18). These data suggest that mouse behavior in the elevated plus-maze is not related to behavior in the forced swimming test and that a forced swimming test before the plus-maze has an anxiogenic effect even after a one-week interval.

Hyperventilation in panic disorder patients and healthy first-degree relatives

Our aim was to observe the induction of panic attacks by a hyperventilation challenge test in panic disorder patients (DSM-IV) and their healthy first-degree relatives. We randomly selected 25 panic disorder patients, 31 healthy first-degree relatives of probands with panic disorder and 26 normal volunteers with no family history of panic disorder. All patients had no psychotropic drugs for at least one week. They were induced to hyperventilate (30 breaths/min) for 4 min and anxiety scales were applied before and after the test. A total of 44.0% (N = 11) panic disorder patients, 16.1% (N = 5) of first-degree relatives and 11.5% (N = 3) of control subjects had a panic attack after hyperventilating (chi² = 8.93, d.f. = 2, P = 0.011). In this challenge test the panic disorder patients were more sensitive to hyperventilation than first-degree relatives and normal volunteers. Although the hyperventilation test has a low sensitivity, our data suggest that there is no association between a family history of panic disorder and hyperreactivity to an acute hyperventilation challenge test. Perhaps cognitive variables should be considered to play a specific role in this association since symptoms of a panic attack and acute hyperventilation overlap.

The brain decade in debate: II. Panic or anxiety? From animal models to a neurobiological basis

This article is a transcription of an electronic symposium sponsored by the Brazilian Society of Neuroscience and Behavior (SBNeC). Invited researchers from the European Union, North America and Brazil discussed two issues on anxiety, namely whether panic is a very intense anxiety or something else, and what aspects of clinical anxiety are reproduced by animal models. Concerning the first issue, most participants agreed that generalized anxiety and panic disorder are different on the basis of clinical manifestations, drug response and animal models. Also, underlying brain structures, neurotransmitter modulation and hormonal changes seem to involve important differences. It is also common knowledge that existing animal models generate different types of fear/anxiety. A challenge for future research is to establish a good correlation between animal models and nosological classification.

Influence of a multideficient diet from northeastern Brazil on resting blood pressure and baroreflex sensitivity in conscious, freely moving rats

The "regional basic diet" or RBD is a multideficient diet (providing 8% protein) which is known to produce dietary deficiencies in some populations in northeastern Brazil. The present study investigated the effects of RBD-induced malnutrition on resting blood pressure and baroreflex sensitivity in conscious rats. Malnourished rats were obtained by feeding dams the RBD during mating and pregnancy (RBD-1 group) or during nursing and a 10-day period after weaning (RBD-2 group). At 90 days of age, only RBD-2 rats weighed significantly (P<0.001) less than control rats born to dams fed a standard commercial diet (23% protein) during pregnancy and nursing. Baseline mean arterial pressure and heart rate of both RBD-1 and RBD-2 rats were comparable to those of controls. The slopes for both reflex bradycardia and tachycardia (bpm/mmHg) induced by intravenous phenylephrine and sodium nitroprusside, respectively, were unchanged in either RBD-1 (-2.08 ± 0.11 and -3.10 ± 0.43, respectively) or RBD-2 (-2.32 ± 0.30 and -3.73 ± 0.53, respectively) rats, when compared to controls (-2.09 ± 0.10 and -3.17 ± 0.33, respectively). This study shows that, after a prolonged period of nutritional recovery, the patterns of resting blood pressure and baroreflex sensitivity of both pre- and postnatally malnourished rats were similar to those of controls. The decreased body weight and the tendency to increased reflex tachycardia in RBD-2 rats may suggest that this type of maternal malnutrition during lactation is more critical than during pregnancy.

Psychometric properties of the Portuguese version of the State-Trait Anxiety Inventory applied to college students: factor analysis and relation to the Beck Depression Inventory

The psychometric properties of the Portuguese version of the trait form of the State-Trait Anxiety Inventory (STAI-T) and its relation to the Beck Depression Inventory (BDI) were evaluated in a large Brazilian college student sample containing 845 women and 235 men. STAI-T scores tended to be higher for women, singles, those who work, and subjects under 30 years. Factor analysis of the STAI-T for total sample and by gender yielded two factors: the first representing a mood dimension and the second being related to worrying or cognitive aspects of anxiety. In order to study the relation between anxiety and depression measures, factor analysis of the combination of the 21 BDI items and the 20 STAI-T items was also carried out. The analysis resulted in two factors that were analyzed according to the tripartite model of anxiety and depression. Most of the BDI items (measuring positive affectivity and nonspecific symptoms of depression) were loaded on the first factor and four STAI-T items that measure positive affectivity. The remaining STAI-T items, all of them measuring negative affect, remained in the second factor. Thus, factor 1 represents a depression dimension and factor 2 measures a mood-worrying dimension. The findings of this study suggest that, although widely used as an anxiety scale, the STAI-T in fact measures mainly a general negative affect.

Effect of acute treatment with a water-alcohol extract of Erythrina mulungu on anxiety-related responses in rats

We investigated the effect of acute oral treatment with a water-alcohol extract of the inflorescence of Erythrina mulungu (EM, Leguminosae-Papilionaceae) (100, 200 and 400 mg/kg) on rats submitted to different anxiety models: the elevated T-maze (for inhibitory avoidance and escape measurements), the light/dark transition, and the cat odor test. These models were selected for their presumed capacity to demonstrate specific subtypes of anxiety disorders as recognized in clinical practice. Treatment with 200 mg/kg EM impaired avoidance latencies (avoidance 1 - 200 mg/kg EM: 18 ± 7 s, control group: 40 ± 9 s; avoidance 2 - 200 mg/kg EM: 15 ± 4 s, control group: 110.33 ± 38 s) in a way similar to the reference drug diazepam (avoidance 1: 3 ± 0.79 s; avoidance 2: 3 ± 0.76 s), without altering escape. Additionally, the same treatments increased the number of transitions (200 mg/kg EM: 6.33 ± 0.90, diazepam: 10 ± 1.54, control group: 2.78 ± 0.60) between the two compartments and the time spent in the lighted compartment in the light/dark transition model (200 mg/kg EM: 39 ± 7 s; diazepam: 61 ± 9 s; control group: 14 ± 4 s). The dose of 400 mg/kg EM also increased this last measurement (38 ± 8 s). These results were not due to motor alterations since no significant effects were detected in the number of crossings or rearings in the arena. Furthermore, neither EM nor diazepam altered the behavioral responses of rats to a cloth impregnated with cat odor. These observations suggest that EM exerts anxiolytic-like effects on a specific subset of defensive behaviors, particularly those that have been shown to be sensitive to low doses of benzodiazepines.

Contrast sensitivity to radial frequencies modulated by Jn and j n Bessel profiles

We measured human contrast sensitivity to radial frequencies modulated by cylindrical (Jo) and spherical (j o) Bessel profiles. We also measured responses to profiles of j o, j1, j2, j4, j8, and j16. Functions were measured three times by at least three of eight observers using a forced-choice method. The results conform to our expectations that sensitivity would be higher for cylindrical profiles. We also observed that contrast sensitivity is increased with the j n order for n greater than zero, having distinct orderly effects at the low and high frequency ends. For n = 0, 1, 2, and 4 sensitivity tended to occur around 0.8-1.0 cpd while for n = 8 and 16 it seemed to shift gradually to 0.8-3.0 cpd. We interpret these results as being consistent with the possibility that spatial frequency processing by the human visual system can be defined a priori in terms of polar coordinates and discuss its application to study face perception.

Low ethanol consumption increases insulin sensitivity in Wistar rats

Several human studies suggest that light-to-moderate alcohol consumption is associated with enhanced insulin sensitivity, but these studies are not free of conflicting results. To determine if ethanol-enhanced insulin sensitivity could be demonstrated in an animal model, male Wistar rats were fed a standard chow diet and received drinking water without (control) or with different ethanol concentrations (0.5, 1.5, 3, 4.5 and 7%, v/v) for 4 weeks ad libitum. Then, an intravenous insulin tolerance test (IVITT) was performed to determine insulin sensitivity. Among the ethanol groups, only the 3% ethanol group showed an increase in insulin sensitivity based on the increase of the plasma glucose disappearance rate in the IVITT (30%, P<0.05). In addition, an intravenous glucose tolerance test (IVGTT) was performed in control and 3% ethanol animals. Insulin sensitivity was confirmed in 3% ethanol rats based on the reduction of insulin secretion in the IVGTT (35%, P<0.05), despite the same glucose profile. Additionally, the 3% ethanol treatment did not impair body weight gain or plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the present study established that 3% ethanol in the drinking water for 4 weeks in normal rats is a model of increased insulin sensitivity, which can be used for further investigations of the mechanisms involved.

Insulin secretion, insulin sensitivity, and hepatic insulin extraction in first-degree relatives of type 2 diabetic patients

To identify early metabolic abnormalities in type 2 diabetes mellitus, we measured insulin secretion, sensitivity to insulin, and hepatic insulin extraction in 48 healthy normal glucose-tolerant Brazilians, first-degree relatives of type 2 diabetic patients (FH+). Each individual was matched for sex, age, weight, and body fat distribution with a person without history of type 2 diabetes (FH-). Both groups were submitted to a hyperglycemic clamp procedure (180 mg/dl). Insulin release was evaluated in its two phases. The first was calculated as the sum of plasma insulin at 2.5, 5.0, 7.5, and 10.0 min after the beginning of glucose infusion, and the second as the mean plasma insulin level in the third hour of the clamp procedure. Insulin sensitivity index (ISI) was the mean glucose infusion rate in the third hour of the clamp experiment divided by the mean plasma insulin concentration during the same period of time. Hepatic insulin extraction was determined under fasting conditions and in the third hour of the clamp procedure as the ratio between C-peptide and plasma insulin levels. FH+ individuals did not differ from FH- individuals in terms of the following parameters [median (range)]: a) first-phase insulin secretion, 174 (116-221) vs 207 (108-277) µU/ml, b) second-phase insulin secretion, 64 (41-86) vs 53 (37-83) µU/ml, and c) ISI, 14.8 (9.0-20.8) vs 16.8 (9.0-27.0) mg kg-1 min-1/µU ml-1. Hepatic insulin extraction in FH+ subjects was similar to that of FH- ones at basal conditions (median, 0.27 vs 0.27 ng/µU) and during glucose infusion (0.15 vs 0.15 ng/µU). Normal glucose-tolerant Brazilian FH+ individuals well-matched with FH- ones did not show defects of insulin secretion, insulin sensitivity, or hepatic insulin extraction as tested by hyperglycemic clamp procedures.

Contrast sensitivity to angular frequency gratings is not higher than to Cartesian gratings

When contrast sensitivity functions to Cartesian and angular gratings were compared in previous studies the peak sensitivity to angular stimuli was reported to be 0.21 log units higher. In experiments carried out to repeat this result, we used the same two-alternative forced-choice paradigm, but improved experimental control and precision by increasing contrast resolution from 8 to 12 bits, increasing the screen refresh rate from 30 Hz interlaced to 85 Hz non-interlaced, linearizing the voltage-luminance relation, modulating luminance in frequencies that minimize pixel aliasing, and improving control of the subject's exposure to the stimuli. The contrast sensitivity functions to Cartesian and angular gratings were similar in form and peak sensitivity (2.4 cycles per visual degree (c/deg) and 32 c/360º, respectively) to those reported in a previous study (3 c/deg and 32 c/360º, respectively), but peak sensitivity to angular stimuli was 0.13 log units lower than that to Cartesian stimuli. When the experiment was repeated, this time simulating the experimental control level used in the previous study, no difference between the peak sensitivity to Cartesian and angular stimuli was found. This result agrees with most current models that assume Cartesian filtering at the first visual processing stage. The discrepancy in the results is explained in part by differences in the degree of experimental control.

Comparison of angular frequency contrast sensitivity in young and older adults

The aim of the present study was to measure contrast sensitivity curves for angular frequencies in the range between 2 and 96 cycles/360º in older human adult volunteers and to compare these measurements with the more usual contrast sensitivity functions for sine-wave gratings. All subjects were free of identifiable ocular disease and had normal acuity. We measured the contrast thresholds for young adults (N = 6; age range, 20-26 years) and older adults (N = 6; age range, 60-67 years) using the psychophysical forced-choice method. In this paradigm the volunteers had to choose the stimulus containing a test frequency at low contrast (e.g., either a sine-wave grating or an angular frequency stimulus), or another neutral stimulus at mean luminance (without any contrast). Older adults presented a loss in contrast sensitivity at high and medium angular frequencies compared to the young adults (i.e., from 8 to 96 cycles/360º). Contrary to expectation, contrast sensitivity at low angular frequencies, i.e., 2 and 4 cycles/360º, was better for the older group than for the younger group. On the other hand, contrast sensitivity for sine-wave gratings at 3 and 4 cpd was higher for young adults as expected. These results suggest age-related changes in the contrast sensitivity function for angular frequencies.

Comparison of the homeostasis model assessment and quantitative insulin sensitivity check index with data from forearm metabolic studies for the in vivo assessment of insulin sensitivity

The present study was designed to compare the homeostasis model assessment (HOMA) and quantitative insulin sensitivity check index (QUICKI) with data from forearm metabolic studies of healthy individuals and of subjects in various pathological states. Fifty-five healthy individuals and 112 patients in various pathological states, including type 2 diabetes mellitus, essential hypertension and others, were studied after an overnight fast and for 3 h after ingestion of 75 g of glucose, by HOMA, QUICKI and the forearm technique to estimate muscle uptake of glucose combined with indirect calorimetry (oxidative and non-oxidative glucose metabolism). The patients showed increased HOMA (1.88 ± 0.14 vs 1.13 ± 0.10 pmol/l x mmol/l) and insulin/glucose (I/G) index (1.058.9 ± 340.9 vs 518.6 ± 70.7 pmol/l x (mg/100 ml forearm)-1), and decreased QUICKI (0.36 ± 0.004 vs 0.39 ± 0.006 (µU/ml + mg/dl)-1) compared with the healthy individuals. Analysis of the data for the group as a whole (patients and healthy individuals) showed that the estimate of insulin resistance by HOMA was correlated with data obtained in the forearm metabolic studies (glucose uptake: r = -0.16, P = 0.04; non-oxidative glucose metabolism: r = -0.20. P = 0.01, and I/G index: r = 0.17, P = 0.03). The comparison of QUICKI with data of the forearm metabolic studies showed significant correlation between QUICKI and non-oxidative glucose metabolism (r = 0.17, P = 0.03) or I/G index (r = -0.37, P < 0.0001). The HOMA and QUICKI are good estimates of insulin sensitivity as data derived from forearm metabolic studies involving direct measurements of insulin action on muscle glucose metabolism.

Contrast sensitivity threshold measured by sweep-visual evoked potential in term and preterm infants at 3 and 10 months of age

Although healthy preterm infants frequently seem to be more attentive to visual stimuli and to fix on them longer than full-term infants, no difference in visual acuity has been reported compared to term infants. We evaluated the contrast sensitivity (CS) function of term (N = 5) and healthy preterm (N = 11) infants at 3 and 10 months of life using sweep-visual evoked potentials. Two spatial frequencies were studied: low (0.2 cycles per degrees, cpd) and medium (4.0 cpd). The mean contrast sensitivity (expressed in percentage of contrast) of the preterm infants at 3 months was 55.4 for the low spatial frequency (0.2 cpd) and 43.4 for the medium spatial frequency (4.0 cpd). At 10 months the low spatial CS was 52.7 and the medium spatial CS was 9.9. The results for the term infants at 3 months were 55.1 for the low spatial frequency and 34.5 for the medium spatial frequency. At 10 months the equivalent values were 54.3 and 14.4, respectively. No difference was found using the Mann-Whitney rank sum T-test between term and preterm infants for the low frequency at 3 or 10 months or for the medium spatial frequency at 3 or 10 months. The development of CS for the medium spatial frequency was equally fast for term and preterm infants. As also observed for visual acuity, CS was equivalent among term and preterm infants, suggesting that visual experience does not modify the development of the primary visual pathway. An earlier development of synapses in higher cortical visual areas of preterm infants could explain the better use of visual information observed behaviorally in these infants.

The relationship between salivary cortisol concentrations and anxiety in adolescent and non-adolescent pregnant women

The main purpose of the present study was to determine the relationship between salivary cortisol concentrations and self-report anxiety in 50 adolescent and 178 non-adolescent women during the last month of pregnancy. The subjects were randomly selected from a previous study involving women who attended antenatal care from September 1997 to August 2000 at 17 health services in Southeast Brazil. Salivary cortisol was measured with an enzyme immunoassay kit, and anxiety was assessed by the State-Trait Anxiety Inventories (STAI) of Spielberger. After saliva collection, the participants completed the STAI. Mean concentrations of cortisol for both pregnant adolescents (14.17 ± 6.78 nmol/l) and non-adolescents (13.81 ± 8.51 nmol/l) were similar (P = 0.89). Forty-three percent of the pregnant adolescents and 30.5% of the non-adolescents felt anxious at the time of being questioned (State Anxiety Inventory (SAI) scores >40; P = 0.06). Cortisol concentrations in adolescents were negatively related to the SAI scores (r = -0.39; P = 0.01) which assess a temporary condition of anxiety. There was a statistically significant difference in mean cortisol concentrations between adolescents with low (<=40) and high (>40) SAI scores (P = 0.03, t-test), but no differences for non-adolescents. The negative relationship between salivary cortisol concentrations and anxiety scores in adolescents may be due to puberty-related hormone differences during this period of life. Pregnant adolescents may possess unique biological or psychological characteristics compared to adults and non-pregnant adolescents. Thus, we need to know more about the hypothalamic-pituitary-adrenocortical axis of pregnant adolescents.