Photoaffinity labeling of the T cell receptor on living cytotoxic T lymphocytes.

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of an antigenic peptide with MHC class I molecules and the TCR on living cells. Two photoreactive derivatives of the H-2Kd (Kd) restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) were used. The first derivative contained an N-terminal photoreactive iodo, 4-azido salicyloyl (IASA) group and biotin on the TCR contact residue Lys259 [IASA-YIPSAEK(biotin)I]. As previously described, this derivative selectively bound to and labeled the Kd molecule. The second photoreactive compound, the isomeric biotin-YIPSAEK(IASA)I, also efficiently bound to the Kd molecule, but failed to label this protein. A CTL clone derived from a mouse immunized with this derivative recognized this conjugate but not the parental P. berghei circumsporozoite peptide or the [IASA-YIPSAEK-(biotin)I] derivative in an Kd-restricted manner. Incubation of the cloned CTL cells with biotin-YIPSAEK(IASA)I, but not its isomer, followed by UV irradiation resulted in photoaffinity labeling of the TCR-alpha chain that was dependent on the conjugate binding to the Kd molecule. The TCR labeling was partially inhibited by anti-LFA 1 and anti-ICAM1 mAb, but was increased by addition of beta 2m or soluble KdQ10. The exquisite labeling selectivity of the two photoprobes opens a new, direct approach to the molecular analysis of antigen presentation and recognition by living CTL.

Evaluation of the 3D symmetry of the knees when walking using na ambulatory inertia system: results 14 months after ACL Plasty.

Purpose of the study: Reconstruction of the anterior cruciate ligament (ACL) controls laxity but does not enable restoration of strictly normal 3D kinematics. The purpose of this study was to compare the kinematics of the pathological knee with that of the healthy knee after ACL plasty. This study applied a new ambulatory system using miniature captors. Material and method: Five patients with an isolated injury of the ACL participated in this study. The patients were assessed after injury (T1), at five months (T2), and at 14 months (T3) after surgery. The assessment included laxity (KT-1000), the IKDC score and the Lysholm score. The 3D angles of the knees were measured when walking 30 m on flat ground using a system composed of to small inertia units (3D accelerometer and 3D gyroscope) and a portable recorder. Functional settings were optimised and validating to ensure easy precise measurement of the 3D angles. Symmetry of the two knees was quantified using a symmetry index (SI) (difference in amplitude normalised in relation to mean amplitude) and the correlation coefficient CC. Results: Clinical indicators improved during the follow-up (IKDC T1: 3C, 2C; T2: 5B; T3: 2A, 3B; subjective IKD: 53-95; Lysholm 67-96). Mean laxity improved from 8.6m to 2.5 mm. The gait analysis showed increased symmetry in terms of amplitude for flexion-extension (SI: −17% at T1, −1% at T2, 1% at T3), and an increase in symmetry in terms of the rotation signature (CC: 0.16 at T1, 0.99 at T2, 0.99 at T3). There was no trend to varus-valgus. Discussion: This study demonstrates the clinical application of the new ambulatory system for measuring 3D angles of the knee joint. Joint symmetry increased after ACL plasty but still showed some perturbation at 14 months. The results observed here are in agreement with the literature. Other patients and other types of gait are being analysed. Conclusion: This portable system allows gait analysis outside the laboratory, before and after ACL injury. It is very useful for follow-up after surgery.

Effects of the α₂-adrenergic agonist clonidine on the pharmacodynamics and pharmacokinetics of 3,4-methylenedioxymethamphetamine in healthy volunteers.

The mechanism of action of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) involves the carrier-mediated and potentially vesicular release of monoamines. We assessed the effects of the sympatholytic α₂-adrenergic receptor agonist clonidine (150 μg p.o.), which inhibits the neuronal vesicular release of norepinephrine, on the cardiovascular and psychotropic response to MDMA (125 mg p.o.) in 16 healthy subjects. The study used a randomized, double-blind, placebo-controlled crossover design with four experimental sessions. The administration of clonidine 1 h before MDMA reduced the MDMA-induced increases in plasma norepinephrine concentrations and blood pressure but only to the extent that clonidine lowered norepinephrine levels and blood pressure compared with placebo. Thus, no interaction was found between the cardiovascular effects of the two drugs. Clonidine did not affect the psychotropic effects or pharmacokinetics of MDMA. The lack of an interaction of the effects of clonidine and MDMA indicates that vesicular release of norepinephrine, which is inhibited by clonidine, does not critically contribute to the effects of MDMA in humans. Although clonidine may be used in the treatment of stimulant-induced hypertensive reactions, the present findings do not support a role for α₂-adrenergic receptor agonists in the prevention of psychostimulant dependence.

Distinction of males of the Lutzomyia intermedia (Lutz & Neiva, 1912) species complex by ratios between dimensions and by an artificial neural network (Diptera: Psychodidae, Phlebotominae)

The females of the two species of the Lutzomyia intermedia complex can be easily distinguished, but the males of each species are quite similar. The ratios between the extra-genital and the genital structures of L. neivai are larger than those of L. intermedia s. s., according to ANOVA. An artificial neural network was trained with a set of 300 examples, randomly taken from a sample of 358 individuals. The input vectors consisted of several ratios between some structures of each insect. The model was tested on the remaining 58 insects, 56 of which (96.6%) were correctly identified. This ratio of success can be considered remarkable if one takes into account the difficulty of attaining comparable results using traditional statistical techniques.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers.

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, cate-cholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

Influence of the blood meal source on the biology of Meccus picturatus Usinger 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood meal and beginning of feeding, feeding time, postfeed defecation delay,life time, mortality and fecundity for each stage of Meccus picturatus, life-cycle were evaluated and compared in two cohorts of M. picturatus fed on hens or rabbits. The hatching rate observed for each of the two studied groups of eggs was 78.1% (n = 2298) on the group fed on hens and 82.1% (n = 2704) on that fed on rabbits, and the average time of hatching was 20 days. Mean time-lapse for beginning feeding was under 3 min in nymphal stages and postfeed defecation delay was under 10 min in all stages, in both cohorts. Mean feeding time was significantly (P < 0.05) shorter in triatomines fed on hens than on rabbits. A similar number of nymphs of each cohort, 69 fed on hens (34.5%) and 68 fed on rabbits (34%), completed the cycle. No significantly (P > 0.05) differences were recorded among the average times from NI to adult in the cohort fed on hens (196.8 ± 15.8 days) and the average time in the cohort fed on rabbits (189.5 ± 22.9). The average span in days for each stage fed on hens was not significantly different to the average span for each stage fed on rabbits. The number of blood meals at each nymphal stage varied from 1 to 6 in both cohorts. The mortality rates were higher on fifth nymphal stage, in both cohorts. No significant (P > 0.05) differences were recorded on mortality rates on most nymphal stages of both cohorts. The average number of eggs laid per female from the cohort fed on hens in a 9-month period was 791.1, whereas the average number of eggs in the cohort fed on rabbits was 928.3.

Monitoring of the dissemination of Salmonella in the chicken Frankfurt-sausage productionline of a sausage factory in the state of São Paulo, Brazil

Poultry meat and its derivatives are among the foodstuffs considered by environmental health authorities to present the highest risks to the public. A total of 185 samples were collected in five monthly batches, from different processing stages in a sausage plant that uses mechanically-deboned chicken meat (MDCM), and testedfor the presence of Salmonella. Enrichment was carried out in both Kauffman's tetrathionate broth and Rappaport-Vassiliadis broth and isolation on Salmonella-Shigella agar and brilliant-green agar. Live Salmonella bacteria were isolated from six samples of the raw meat and from the emulsion, in batches three, four, and five, but not from any sample in batches one or two. The six isolated strains were all classified as Salmonella Albany, which has not previously been reported in MDCM. Of the two enrichment broths, Rappaport-Vassiliadis gave the better results. The pattern of contamination suggests a probable common source, given that a new supplier was used in the third, fourth, and fifth months. It was also shown that the industrial cooking was effective in preventing Salmonella surviving in the final product.

The "help" question doesn't help when screening for major depression: external validation of the three-question screening test for primary care patients managed for physical complaints.

BACKGROUND: Major depression, although frequent in primary care, is commonly hidden behind multiple physical complaints that are often the first and only reason for patient consultation. Major depression can be screened by two validated questions that are easier to use in primary care than the full DSM-IV criteria. A third question, called the "help" question, improves the specificity without apparently decreasing the sensitivity of this screening procedure. We validated the abbreviated screening procedure for major depression with and without the "help" question in primary care patients managed for a physical complaint. METHODS: This diagnostic accuracy study used data from a cohort study called SODA (for SOmatisation Depression Anxiety ) conducted by 24 general practitioners (GPs) in western Switzerland that included patients over 18 years of age with at least one physical complaint at index consultation. Major depression was identified with the full Patient Health Questionnaire. GPs were asked to screen patients for major depression with the three screening questions one year after inclusion. RESULTS: Out of 937 patients with at least one physical complaint, 751 were eligible one year after index consultation. Major depression was diagnosed in 69/724 (9.5%) patients. The sensitivity and specificity of the two-question method alone were 91.3% (95% confidence interval 81.4-96.4%) and 65.0% (95% confidence interval 61.2-68.6%), respectively. Adding the "help" question decreased the sensitivity (59.4% ; 95% confidence interval 47.0-70.9%) but improved the specificity (88.2% ; 95% confidence interval 85.4-90.5%) of the three-question method. CONCLUSIONS: The use of two screening questions for major depression was associated with high sensitivity and low specificity in primary care patients presenting a physical complaint. Adding the "help" question improved the specificity but clearly decreased the sensitivity; when using the "help" question; four out of ten patients with depression will be missed, compared to only one out of ten with the two-question method. Therefore, the "help" question is not useful as a screening question, but may help discussing management strategies.

Reconstitution into liposomes of the tonoplast ATP-and pyrophosphate-dependent proton pumps from Rubus hispidus cell cultures.

The ATP- and pyrophosphate-dependent proton pumps from tonoplast-enriched vesicles prepared from Rubus hispidus cell cultures were solubilized in the presence of polyoxyethylene(9,10)p-t-octylphenol (Triton X-100) and reconstituted into liposomes of soybean phospholipids, using Bio-Beads SM-2 to remove the detergent. The specific activity of the two pumps was greatly increased by the solubilization-reconstitution procedure. Identical characteristics were found for pyrophosphate-dependent proton transport in native and reconstituted vesicles. On the other hand, the ATP-dependent proton transport of the reconstituted vesicles was no longer inhibited by KNO3.

A new perspective on the zoogeography of the sibling mouse-eared bat species Myotis myotis and Myotis blythii: Morphological, genetical and ecological evidence

The actual geographic distribution of the two sibling mouse-eared bat species Myotis myotis and Myotis blythii, which occur widely sympatrically in the western Palaearctic region, remains largely controversial. This concerns particularly the specific attribution of marginal populations from the Mediterranean islands and from adjacent areas of North Africa and Asia, which are morphologically intermediate between continental M. myotis and M. blythii from Europe. This study attempts to clarify this question by using four different approaches: cranial morphology, external morphology, genetics and trophic ecology. The three latter methods show unambiguously that North Africa, Malta, Sardinia and Corsica are presently inhabited by monospecific populations of M. myotis. In contrast, cranial morphometrics do not yield conclusive results. These results contradict all recent studies, which attribute North African and Maltese mouse-eared bats to M. blythii and consider that Sardinia and Corsica harbour sympatric populations of the two species. As concerns south-eastern populations, doubts are also expressed about the attribution of the subspecific taxon omari which may actually refer to M. myotis instead of M. blythii. Protein electrophoresis is presently the only absolute method available for determining M. myotis and M. blythii throughout their distribution ranges. However, species identification may be approached by relying on less sophisticated morphometrical methods as presented in this study. Species-specific habitat specializations are probably responsible for the differences observed between the geographic distributions of M. myotis and M. blythii, as they provide a logical groundwork for a coherent model of speciation for these two bat species.

The hierarchical structures of the NEO-PI-R and the 16PF5

The present study compares the higher-level dimensions and the hierarchical structures of the fifth edition of the 16 PF with those of the NEO PI-R. Both inventories measure personality according to five higher-level dimensions. These inventories were however constructed according to different methods (bottom-up vs. top-down). 386 participants filled out both questionnaires. Correlations, regressions and canonical correlations made it possible to compare the inventories. As expected they roughly measure the same aspects of personality. There is a coherent association among four of the five dimensions measured in the tests. However Agreeableness, the remaining dimension in the NEO PI-R, is not represented in the 16 PF 5. Our analyses confirmed the hierarchical structures of both instruments, but this confirmation was more complete in the case of the NEO PI-R. Indeed, a parallel analysis indicated that a four-factor solution should be considered in the case of the 16 PF 5. On the other hand, the NEO PI-R's five-factor solution was confirmed. The top-down construction of this instrument seems to make for a more legible structure. Of the two five-dimension constructs, the NEO PI-R thus seems the more reliable. This confirms the relevance of the Five Factor Model of personality.

Immune response to the mumps component of the MMR vaccine in the routine of immunisation services in the Brazilian National Immunisation Program

A non-controlled longitudinal study was conducted to evaluate the combined vaccine against measles, mumps and rubella (MMR) immunogenicity in 150 children vaccinated in the routine of three health units in the city of Rio de Janeiro, Brazil, 2008-2009, without other vaccines administered during the period from 30 days before to 30 days after vaccination. A previous study conducted in Brazil in 2007, in 1,769 children ranging from 12-15 months of age vaccinated against yellow fever and MMR simultaneously or at intervals of 30 days or more between doses, had shown low seroconversion for mumps regardless of the interval between administration of the two vaccines. The current study showed 89.5% (95% confidence interval: 83.3; 94.0) seroconversion rate for mumps. All children seroconverted for measles and rubella. After revaccination, high antibody titres and seroconversion rates were achieved against mumps. The results of this study and others suggest that two MMR doses confer optimal immunoresponses for all three antigens and the possible need for additional doses should be studied taking into account not only serological, but also epidemiological data, as there is no serological correlate of protection for mumps.

In vitro activity of the hydroethanolic extract and biflavonoids isolated from Selaginella sellowii on Leishmania (Leishmania) amazonensis

This study is the first phytochemical investigation of Selaginella sellowii and demonstrates the antileishmanial activity of the hydroethanolic extract from this plant (SSHE), as well as of the biflavonoids amentoflavone and robustaflavone, isolated from this species. The effects of these substances were evaluated on intracellular amastigotes of Leishmania (Leishmania) amazonensis, an aetiological agent of American cutaneous leishmaniasis. SSHE was highly active against intracellular amastigotes [the half maximum inhibitory concentration (IC50) = 20.2 µg/mL]. Fractionation of the extract led to the isolation of the two bioflavonoids with the highest activity: amentoflavone, which was about 200 times more active (IC50 = 0.1 μg/mL) and less cytotoxic than SSHE (IC50 = 2.2 and 3 μg/mL, respectively on NIH/3T3 and J774.A1 cells), with a high selectivity index (SI) (22 and 30), robustaflavone, which was also active against L. amazonensis (IC50 = 2.8 µg/mL), but more cytotoxic, with IC50 = 25.5 µg/mL (SI = 9.1) on NIH/3T3 cells and IC50 = 3.1 µg/mL (SI = 1.1) on J774.A1 cells. The production of nitric oxide (NO) was lower in cells treated with amentoflavone (suggesting that NO does not contribute to the leishmanicidal mechanism in this case), while NO release was higher after treatment with robustaflavone. S. sellowii may be a potential source of biflavonoids that could provide promising compounds for the treatment of cutaneous leishmaniasis.

Habitat, morphology and karyotype of the Saharan shrew Crocidura tarfayaensis (Mammalia : Soricidae)

The Saharan shrew Crocidura tarfayaensis Vesmanis and Vesmanis, 1980, has a limited disribution along the Atlantic coast of Sahara, south of Agadir (Morocco) through Western Sahara into Mauritania and is only known from few captures and some owl pellets. Here we report field data from the successful trapping of five specimens of C. tarfayaensis in the Guelmim region. The habitat was characterized by sand dunes along a river, with dense shrubberies of Tamarix sp., the huge grass Erianthus ravennae (Poaceae) and flat bushes of Atriplex glauca var. ifniensis (Chenopodiaceae). Morphological discrimination with C. whitakeri were examined. The chromosomes of C. tarfayaensis revealed a karyotype of 2n = 36, similar to that of the Canary shrew C. canariensis and the Sicilian shrew C. sicula. In conclusion, C. tarfayaensis seems to be a descendant of the presumed continental ancestor of the two island species.

Genetic differentiation of disjunct populations of the ants Formicaaquilonia and Formica lugubris in Europe.

The species Formica aquilonia and F. lugubris of the mound-building red wood ants have a disjunct boreoalpine distribution in Europe. The populations of F. aquilonia in Finland, Switzerland and the British Isles show little genetic differentiation, whereas the populations of F. lugubris show considerable differentiation. The Central European populations morphologically identified as F. lugubris can be genetically divided into two groups (here called types A and B). Type B is found in the Alps and the Jura mountains, and is genetically inseparable from F. aquilonia. Type A lives sympatrically with type B in the Jura mountains and is also found in the British Isles. Sympatry of the two types in the Jura shows that these are separate species. It remains open whether type B is morphologically atypical F. aquilonia or whether it is a separate species, perhaps with a past history of introgression between F. aquilonia and F. lugubris. The gene frequencies in the Finnish populations of F. lugubris differ from those of both types A and B. Genetic differences within F. lugubris indicate that the populations have evolved separately for a long time. The social structure of F. lugubris colonies also shows geographic variation. The nests in Finland and the British Isles seem to be mainly monogynous and monodomous, whereas the nests in Central Europe are polygynous and form polydomous colonies. F. aquilonia has polygynous and polydomous colonies in all populations studied.