Development of a PCR-based tool for detecting immunologically relevant Alt a 1 and Alt a 1 homologue coding sequences.

Alt a 1 has been recognized as the most important allergen produced by the Pleosporaceae family and is a risk factor for asthma development and/or exacerbation. The aim of this study was to develop a detection tool that is highly specific for species that produced airborne Alt a 1. Based on the highly conserved internal nucleotide region of the several Alt a 1 sequences that are available in GenBank, a pair of primers (Alta1CF/Alta1CR) was designed. A set of primers used by other authors for the production of recombinant Alt a 1 (A21F/A21R) was also tested. The molecular analyses were based on the polymerase chain reaction (PCR) amplification and sequencing of the cDNA that was obtained from thirteen common fungal species. The PCR system that utilized Alta1CF/Alta1CR was highly specific, sensitive, and was able to detect an amplicon of approximately 180 bp from Alt a 1 and Alt a 1-like encoding genes from A. alternata, A. tenuissima, A. infectoria, U. botrytis, and S. botryosum. In contrast, the A21F/A21R primers were specific for the very close taxonomically related species A. alternata and A. tenuissima. Thus, this rapid, sensitive, and specific detection tool can be used to assess Alt a 1 exposure levels and to inform the implementation of the appropriate public health measures.

Evaluation and comparison of commercially available latex extracts for skin prick tests.

BACKGROUND: Crude latex extracts are commonly used in skin prick tests (SPT) for the diagnosis of natural rubber latex (NRL) allergy. Nevertheless, variations in protein and allergen composition between latex extracts from different manufacturers can hamper a correct diagnosis. OBJECTIVES: To analyze the heterogeneity of proteins and allergens in latex extracts from 7 different manufacturers and to assess its relevance in the diagnosis of latex allergy. METHODS: Seven latex SPT extracts were analyzed for protein content using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The 4 major allergens Hev b 1, Hev b 3, Hev b 5, and Hev b 6.02 were also quantified using enzyme immunoassay. All commercial extracts were tested for their in vitro allergenic capacity using microarray inhibition assays and for their ability to induce biological reactivity in latex-allergic patients undergoing SPT. RESULTS: The protein content of the extracts varied widely from 8.0 microg/mL to 526.5 microg/mL. SDS-PAGE revealed broad differences in protein profiles between the extracts. Marked variability in the contents of all 4 major allergens was observed, and Hev b 3 and Hev b 5 were undetectable in some extracts. Microarray inhibition assays and SPT demonstrated relevant differences in allergenic capacity between the extracts. CONCLUSIONS: The marked heterogeneity in protein and allergen content of latex extracts from different manufacturers could explain the broad spectrum of SPT results recorded. Our findings suggest that the extracts used for the diagnosis of latex allergy should be improved and standardized.

Different in vivo reactivity profile in health care workers and patients with spina bifida to internal and external latex glove surface-derived allergen extracts.

BACKGROUND: Allergy to natural rubber latex is a well-recognized health problem, especially among health care workers and patients with spina bifida. Despite latex sensitization being acquired in health institutions in both health care workers and patients with spina bifida, differences in allergen sensitization profiles have been described between these two risk groups. OBJECTIVE: To investigate the in vivo reactivity of health care workers and patients with spina bifida to extracts of internal and external surfaces of latex gloves and also to specific extracts enriched in major allergens for these risk groups. METHODS: Gloves from different manufacturers were used for protein extraction, and salt precipitation and hydrophobic interaction chromatography (HIC) were applied to obtain the enriched latex extracts. The major latex allergens were quantified by an enzyme immunoassay. The extracts obtained were tested in 14 volunteers using skin prick tests (SPT). RESULTS: Latex glove extracts enriched in the hydrophobic allergens that are most often seen in patients with spina bifida were obtained by selective precipitation, whereas HIC produced extracts enriched in the hydrophilic allergens commonly found in health care workers. The health care workers had positive SPTs to glove extracts from internal surfaces and to the hydrophilic allergen-enriched extracts. By contrast, patients with spina bifida had larger skin reactions both to external glove extracts and to the extracts enriched with the hydrophobic major allergens for this risk group. Despite the protein concentration of these extracts being less than half the concentration of the commercial extract, the weal-and-flare reactions were of similar magnitude. CONCLUSION: Using novel latex extracts, our study showed a different in vivo reactivity pattern in health care workers and in patients with spina bifida to extracts of the internal and external surfaces of gloves, which suggests that sensitization may occur by different routes of exposure, and that this influences the allergen reactivity profiles of these risk groups.

Specific berenil-DNA interactions: an approach for separation of plasmid isoforms by pseudo-affinity chromatography.

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.

Latex allergy: new insights to explain different sensitization profiles in different risk groups.

BACKGROUND: Differences in latex allergen sensitization profiles have been described between children subjected to repetitive surgical interventions and health care workers (HCW). 'Major' allergens for patients with spina bifida are Hev b 1, 3 and 7, while for HCW, 'major' allergens are Hev b 2, 5, 6.01 and 13. The reason for these differential sensitization profiles is currently unknown. OBJECTIVES: To investigate latex allergen profiles on internal and external surfaces of natural rubber latex gloves. METHODS: Eighty-two samples of commonly used surgical gloves (41 glove brands) were used for analysis. Specific allergen levels of Hev b 1, 3, 5 and 6.02 on both surfaces of the gloves were quantified using an enzyme immunometric assay, a FITkit (FIT Biotech, Tampere, Finland). RESULTS: Differences in allergen levels were observed between internal and external surfaces of all glove types. Concentrations of Hev b 1 and Hev b 3 were significantly higher on external surfaces, while internal surfaces had higher allergen levels of Hev b 5 and Hev b 6.02. Analysis of surgical and examination gloves, powdered and nonpowdered gloves also showed that the content of Hev b 5 and Hev b 6.02 was significantly higher on internal surfaces while that of Hev b 1 and Hev b 3 was higher on external surfaces. CONCLUSIONS: Our study showed different allergen profiles on internal and external surfaces of natural rubber latex gloves. These results may suggest a relationship between latex allergen localization and sensitization routes in different risk groups.

Changes in experimental conditions alter anti-predator vigilance and sequence predictability in captive marmosets.

Anti-predator vigilance and its sequence predictability in captive adult male black tufted-ear marmosets (Callithrix penicillata) was analyzed under three experimental conditions: (1) four novel (maze) environment habituation trials; (2) six taxidermized (cat) predator confrontation trials in the same maze environment; and (3) four additional maze-only trials, in the absence of the cat stimulus. Significant quantitative and qualitative differences (aerial vs. terrestrial) were observed in scan and glance behaviors within and between the three experimental conditions. Furthermore, inter-scan bout sequence significantly deviated from randomness during the initial maze habituation and predator confrontation trials, as well as during all predator removal trials. This parameter, however, followed a random pattern during the course of the remaining sessions. Therefore, vigilance in marmosets seems to be an important and highly organized component of this species' anti-predation repertoire, inasmuch as it occurs at high rates, alters according to specific environmental cues and has a subtle differential adaptive response after repeated trials.

Bacteriocin production of the probiotic Lactobacillus acidophilus KS400.

In the last years, the use of probiotics, including Lactobacillus species, has received much attention to prevent and treat vaginal disorders. These species have been described as having the ability to colonize the epithelial surface and produce antimicrobial metabolites that are able to control the remaining vaginal microflora. This study aimed to identify and characterize, for the first time, a bacteriocin natively produced by Lactobacillus acidophilus KS400 (probiotic strain from Gynoflor®-Medinova AG, Switzerland) and its antimicrobial activity against relevant urogenital pathogens. After organic acids and hydrogen peroxide neutralization in the fermented Lactobacillus acidophilus KS400 culture medium, bacteriocin activity was tested against the indicator microorganism Lactobacillus delbrueckii ATCC9649. The fermentation of Lactobacillus acidophilus KS400 for bacteriocin production was carried out in batch mode, and its antimicrobial activity, optical density and pH were monitored. After production and extraction, the bacteriocin molecular weight was estimated by electrophoresis and tested against vaginal pathogenic microorganisms. As described for other bacteriocins, batch fermentation profiles indicated that bacteriocin production occurs during the exponential growth phase of the lactobacilli, and declines during their stationary growth phase. The molecular weight of the bacteriocin is approximately 7.5 kDa. The bacteriocin containing protein extract was shown to inhibit the growth of Gardnerella vaginalis, Streptococcus agalactiae, Pseudomonas aeruginosa and the indicator strain Lactobacillus delbrueckii ATCC9649. We conclude that L. acidophilus KS400 produces bacteriocin with antimicrobial activity against relevant urogenital pathogens.

Evidence for hemispheric specialization in the marmoset (Callithrix penicillata) based on lateralization of behavioral/neurochemical correlations.

A correlative study between behavioral, neurochemical and hormonal measures was conducted on male black tufted-ear marmoset monkeys (Callithrix penicillata). Behavioral analysis was performed in order to examine the effects of confrontation with a natural predator (taxidermized oncilla cat, Felis tigrina). The subjects were subjected to four trials without predator, six confrontation trials with predator present, and four trials with the predator removed. Handedness was analyzed by the frequency with which they performed scratching, grooming and hanging behaviors with the left or right hands. The animals' brains were subjected to ex vivo neurochemical analysis of several structures from both hemispheres. The content of monoamines, acetycholine and metabolites were analyzed by HPLC-ED. Plasma levels of cortisol and adrenocorticotrophic hormone (ACTH) were analyzed by chemoluminescence immunoassay. Testosterone plasma concentration was determined by radioimmunoassay. Higher levels of dopamine and acetylcholine were detected in the right caudate/putamen, in comparison to the left. For the remaining areas, similar levels were observed in both hemispheres. A hand preference between and within the behaviors scored was not detected. However, correlative analyses revealed complex interactions between the behavioral and neurochemical measures, particularly in the left hemisphere. Lateralized correlations were found in relation to brain site, type of behavior, neurochemical parameter and treatment condition, thus providing evidence for functional brain asymmetries in this species. Interhemispheric comparisons of neurochemical/behavioral correlations appear to be a promising approach towards delineating hemispheric specialization of functions in this, and perhaps, other species.

[The role played by the autonomic nervous system in the relation between depression and cardiovascular disease]. / Papel del sistema nervioso autónomo en la relación entre depresión y enfermedad cardiovascular.

INTRODUCTION: Findings from several epidemiological studies have revealed that major depression is associated with an increased risk of developing cardiovascular diseases (CVD) and presenting complications and new events in subjects with already-established CVD. The pathophysiological mechanisms responsible for this increased cardiovascular risk in major depression remain unclear. DEVELOPMENT: The aim of this work is to review the literature on the possible pathophysiological mechanisms involved in the relation between major depression and CVD, with special emphasis on the studies dealing with cardiovascular autonomic dysfunction and heart rate variability. Likewise, recent hypotheses concerning the neural mechanisms underlying autonomic dysfunction in subjects with major depression are also discussed. CONCLUSIONS: The evidence that is currently available allows us to hypothesise that there are anomalies in the functioning of the central autonomic neural network in subjects with major depression, and more specifically in the hippocampus, prefrontal cortex and the brain stem nuclei. Such abnormalities, in association with lower central levels of serotonin give rise to a predominance of the sympathetic flow and a loss of cardiac vagal tone. The resulting cardiovascular autonomic dysfunction could be the main cause of the increased cardiovascular risk observed in major depression. In the future, studying the autonomic nervous system may be a useful tool in the development of new therapeutic strategies aimed at reducing cardiovascular morbidity and mortality in subjects with depression.

Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith.

Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.

Alt a 15 is a new cross-reactive minor allergen of Alternaria alternata.

Alternaria alternata is one of the most common saprophytes worldwide that is clinically and epidemiologically associated with severe asthma. Therefore, the identification and characterization of all A. alternata allergens are of major clinical importance. This study describes a new cross-reactive A. alternata allergen that was officially named Alt a 15 by the official Allergen Nomenclature Subcommittee. The complete coding region for Alt a 15 was amplified using 5' and 3' rapid amplification of cDNA ends and PCR. The recombinant protein was produced in Escherichia coli as a 65-kDa fusion protein, and the protein sequence exhibits high homology with several important fungal allergens. Immunoblotting analyses revealed that IgE antibodies from A. alternata-sensitized patients (n=59) bound to rAlt a 15 with a prevalence of 10.2%. All patients who presented sIgE to rAlt a 15 were apparently poly-sensitized to A. alternata and C. lunata. The extensive cross-reactivity between A. alternata and C. lunata serine proteases was confirmed using immunoblotting inhibition assays. Overall, Alt a 15 is an important new cross-reactive allergen of A. alternata that explains some allergies to A. alternata without Alt a 1 sensitization and initial diagnostic errors for allergies to Alternaria. This molecule may improve the accuracy of the diagnosis, the understanding, and the management of IgE-mediated fungal diseases.

High versus low fat/sugar food affects the behavioral, but not the cortisol response of marmoset monkeys in a conditioned-place-preference task.

The effect of a high (chocolate) versus low fat/sugar (chow) food on a conditioned-place-preference (CPP) task was evaluated in marmoset monkeys. Anxiety-related behaviors and cortisol levels before and after the CPP task were also measured. Subjects were habituated to a two-compartment CPP box and then, on alternate days, had access to only one compartment during daily 15-min conditionings, for a total of 14 trials. Marmosets were provisioned with chocolate chips in the CC-paired compartment on odd-numbered trials and standard chow in the CW-paired compartment on even-numbered trials. They were then tested for preferring the CC-paired context after a 24-h interval. During the conditioning, a significantly greater amount (in kcal/trial) of chocolate was consumed than chow, yet the foraging pattern of both food types was similar. On the test trial, the time spent in the CC-paired context increased significantly compared to pre-CPP levels, yet this response was not readily predicted by baseline behavioral or cortisol levels. Also, the chocolate CPP response was positively correlated with foraging time, rather than the amount of calories consumed. The sudden absence of the food increased exploration, while the chocolate CPP effect was associated with vigilance - both anxiety-related behaviors in marmosets. This behavioral profile occurred regardless of any concomitant change or correlation with cortisol. Therefore, the high fat/sugar food was more prone to be overly consumed by the marmosets, to induce a CPP response and to lead to anxiety-related behavior in its absence.

Facilitation of memory by peripheral administration of substance P and naloxone using avoidance and habituation learning tasks.

This paper summarizes results of a series of experiments dealing with the effects of the neuropeptide substance P (SP) on avoidance learning and habituation. Several doses of SP (0.5, 5, 50, 100, 250, 500 micrograms/kg) were administered posttrial intraperitoneally (IP). Three inhibitory one-trial avoidance tasks were used; uphill, step-down and step-through (alcove). Habituation was measured in an open field by recording the number of rearings. The posttrial injection of SP facilitated avoidance responses as well as reduced rearing in a dose- and time-dependent way. Pretraining and pretest injections (IP) of naloxone facilitated avoidance behavior and potentiated the action of SP, also in a dose-dependent manner. These results demonstrate that: a) peripheral posttraining administration of SP enhances memory; b) SP facilitates not only aversive or positively motivated learning tasks, but also habituation, which is a form of learning that involves neither positive nor negative reinforces; c) SP does not exert its effect by a long-lasting proactive action on performance during the testing trial; d) naloxone potentiates the SP posttraining effect. These data, therefore, suggest that memory-enhancing effects of SP are, at least in part, mediated via interactions between this peptide and endogenous opioid systems.

L-DOPA and psychosis: evidence for L-DOPA-induced increases in prefrontal cortex dopamine and in serum corticosterone.

L-DOPA can often induce psychotic reactions during treatment for Parkinson's disease. This study was undertaken to assess, in an animal model of Parkinson's disease, the impact of L-DOPA treatment on two potential biological risk factors for psychosis, namely, an increase in prefrontal cortex dopamine and an increase in the stress-related hormone corticosterone. Hemiparkinsonian rats with unilateral 6-hydroxydopamine (6-OHDA) lesions which resulted in severe unilateral denervation of dopamine neurons were treated with either saline or 25 mg/kg L-DOPA methyl ester (with 2 mg/kg carbidopa). Serum L-DOPA concentrations were found to be positively and highly correlated with serum corticosterone, with medial prefrontal cortex dopamine and with the dopamine metabolite homovanillic acid. Serum L-DOPA, however, was found not to be correlated with serum or brain concentrations of serotonin, 5-hydroxyindoleacetic acid, or norepinephrine. These findings support the possibility that chronic L-DOPA treatment can expose parkinsonian patients to two significant risk factors for psychosis: 1) increased levels of prefrontal cortex dopamine, and 2) increased levels of serum corticosterone.

Increased training in an aversively motivated task attenuates the memory-impairing effects of posttraining N-methyl-D-aspartate-induced amygdala lesions.

This study was designed to examine the effect of variations in the amount of preoperative training on the retention deficit produced by posttraining lesions of the amygdaloid complex (AC). Rats received 1, 10, or 20 training trials in a footshock-motivated escape task 7 days before receiving N-methyl-D-aspartate lesions of the AC. Inhibitory avoidance retention performance, which was measured 4 days postoperatively, indicated that increased training improved retention in AC-lesioned animals as well as in control animals. The retention performance of AC-lesioned animals was impaired when compared with that of controls; however, the impairment was partially attenuated by increased preoperative training. The finding that AC-lesioned animals displayed greater locomotor activity on the retention test compared with nonlesioned controls suggests that the increased activity may have contributed to the impaired inhibitory avoidance retention performance. Two days after the retention test, some of the AC-lesioned animals were subsequently trained on a continuous multiple-trial inhibitory avoidance response in the same apparatus. AC lesions did not block acquisition or retention of the task. These findings suggest that the amygdala may not be a critical site for the permanent changes mediating stimulus-affect associations based on extensive training.

NMDA antagonist effects on the development of L-dopa behavioral sensitization in rats.

This study, which used an animal model of Parkinsonism, evaluated whether the NMDA antagonist MK-801 can prevent the development of L-3-4-dihydroxyphenylalanine (L-DOPA) sensitization. In separate groups, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions were treated with saline, 25 mg/kg L-DOPA methyl ester, 0.1 mg/kg MK-801, or MK-801 plus L-DOPA once per day for 13 days beginning 18 to 20 hr postoperatively, well before the onset of denervation supersensitivity. Following 14 days of withdrawal, all treatment groups were given a saline test and on the next day, an L-DOPA challenge test. Contralateral rotation, the behavioral index of denervation supersensitivity, emerged on Day 7 in both L-DOPA groups. However, on the L-DOPA challenge test, only the L-DOPA group showed enhanced contralateral rotations compared with a drug-naive group. In contrast, the MK-801 and MK-801/L-DOPA groups were indistinguishable from the drug-naive L-DOPA-treated rats. These findings indicate that although MK-801 treatment did not prevent the development of behavioral sensitization to the L-DOPA treatment, it did prevent its persistence following drug withdrawal.

'Priming' enhances the relationship of striatal L-dopa concentration to behavior.

Rats with unilateral 6-hydroxydopamine (6-OHDA) lesions were exposed to L-DOPA treatments, 25 mg kg-1 L-DOPA methyl ester/2 mg kg-1 carbidopa, or to saline. Fourteen days later, both sets of animals were tested with the L-DOPA/carbidopa treatment. The L-DOPA pre-exposed animals exhibited behavioral sensitization as indexed by a higher frequency of contralateral rotations. Although striatal L-DOPA and HVA concentrations were equivalent in the two groups, the L-DOPA treatment induced a 10 fold variation in the range of L-DOPA concentrations in the 6-OHDA striatum in both sets of animals. Importantly, significant correlations between striatal L-DOPA concentration and behavior were obtained only for the L-DOPA pre-exposed animals.

Comparison of neurokinin SP with diazepam in effects on memory and fear parameters in the elevated T-maze free exploration paradigm.

The elevated T-maze was combined with a free exploration protocol, which, in contrast to the conventional procedure, dispenses with handling of the animals during the experimental sessions. This allows measurement of fear indexes derived from the elevated plus-maze as well as assessment of acquisition of open arm avoidance and open arm escape in one continuous session. Retention of the different fear-responses is measured 72 h later without drug treatment. In order to assess the effects of two known anxiolytics in this paradigm, rats received an IP injection of diazepam (1 to 4 mg/kg), substance P (5 to 500 microg/kg) or vehicle (1 ml/kg) and were tested on the T-maze for 5 min. Diazepam elevated open arm activity, indicative of an anxiolytic effect. The drug also increased the latency to escape from the open arms, but did not significantly affect acquisition of open arm avoidance. During the retention trial, diazepam in higher doses impaired the performance of both fear-responses, suggestive of an anterograde amnesic effect. Substance P did not influence acquisition and retention of open arm avoidance and escape. However, in high doses, the peptide increased the sojourn time in the central arena of the maze, indicating reduced fear and, hence, a dissociation between anxiolytic and amnesic effects. The present findings demonstrate that the elevated T-maze free exploration paradigm is sensitive to anxiolytic and memory-modulating effects of drugs.

Substance P and its role in neural mechanisms governing learning, anxiety and functional recovery.

The neurokinin Substance P (SP) is widely distributed in the central nervous system and has been extensively studied in various functional aspects. This review focuses on the behavioral relevance of SP. Here we show that SP can have memory-promoting, reinforcing and anxiolytic-like effects when administered systemically or into the nucleus basalis of the ventral pallidum. These effects seem to be mediated via the SP-preferring NK(1)receptor and differentially related to N- versus C-terminal fragments of the undecapeptide. Secondly, SP injection into the ventral pallidum can lead to increases of acetylcholine in frontal cortex and dopamine in nucleus accumbens, suggesting that the hypermnestic, positively reinforcing and anxiolytic effects observed upon basal forebrain injection of SP are mediated by activation of the nucleus accumbens-ventral pallidum circuitry. Furthermore, SP and certain SP-fragments may not only be considered to have beneficial behavioral effects in normal animals, but can also prevent lesion-induced functional deficits and improve the speed of recovery. This indicates that SP agonists might also have a neuroprotective capacity in parallel with recovery-promoting actions.