Melanin and pyomelanin in Aspergillus fumigatus: from its genetics to host interaction.

Aspergillus fumigatus is a worldwide-distributed saprophytic fungus and the major cause of invasive aspergillosis. This fungus can produce two types of melanin-dihydroxynaphthalene melanin (DHN-melanin) and pyomelanin. These pigments are considered important resistance mechanisms to stress, as well as virulence factors. The aim of this review is to present the current knowledge of the genetic basis and metabolic pathways of melanin production, their activation, function, and interaction with the host immune system. The DHN-melanin pathway is encoded in a cluster that includes six genes (abr1, abr2, ayg1, arp1, arp2, and pksP/alb1 genes) whose encoded proteins seem to be the origin of the pigment in endosomes. These vesicles are secreted and the pigment is subsequently located in the wall of the conidium beneath the rodlet layer. Unlike DHN-melanin, pyomelanin does not have its own biosynthetic pathway but is related to the activation of the L-tyrosine/L-phenylalanine degradation pathway that includes a cluster of six genes (hppD, hmgX, hmgA, fahA, maiA, and hmgR). Its production is due to the polymerization of homogentisic acid and is linked to conidial germination. Despite the knowledge gained in recent years, further studies will be necessary to confirm the pathways that produce these pigments and their role in the virulence mechanisms of A. fumigatus.

Results of Infrainguinal Bypass in Acute Limb Ischaemia.

OBJECTIVE/BACKGROUND: To assess the outcomes of infrainguinal bypass performed for acute limb ischaemia, as well as the predictors of patency, mortality, and amputation. METHODS: This was a retrospective cohort study of patients undergoing infrainguinal bypass between 1998 and 2014. The cohort was stratified according to the indication for surgery into two groups: group A (acute limb ischaemia) and group B (chronic lower extremity ischaemia). Comparative analysis was performed on comorbidities, surgical technique, and outcomes, as well as prognostic factors in group A. RESULTS: In total, 702 bypasses were performed (group A, n = 107; group B, n = 595). Differences between groups were detected in age (65.9 vs. 70.9 years; p = .03), diabetes (16% vs. 49%; p < .01), renal insufficiency (6% vs. 13%; p = .05), stroke (7% vs. 14%; p = .04), and coronary artery disease (13% vs. 28%; p < .01). Patients with acute limb ischaemia more often required general anaesthesia (47% vs. 12%; p < .01) and a short bypass was more often performed (32% vs. 7%; p < .01). Median follow up was 23 and 24 months for groups A and B, respectively. No differences were found in patency rates at 1, 12, and 24 months between groups, but group B had a higher re-intervention rate during follow up. Primary patency in group A was 84%, 63%, and 58%, and in group B it was 88%, 62%, and 53% at 1, 12, and 24 months, respectively (p = .77). Assisted primary patency in group A was 85%, 72%, and 67%, and in group B it was 90%, 74%, and 66% at 1, 12, and 24 months, respectively (p = .61). Secondary patency in group A was 90%, 78%, and 75%, and in group B it was 94%, 80%, and 74% at 1, 12, and 24 months, respectively (p = .80). The freedom from re-intervention rate in group A was 91%, 74%, and 68%, and in group B it was 92%, 76%, and 71%, respectively (p = .04). Acute limb ischaemia was an independent risk factor for amputation (odds ratio [OR] 4.96, 95% confidence interval [CI] 1.74-14.09; p < .01) and mortality (OR 4.13, 95% CI 1.53-11.14; p = .01) at 30 days. In group A, female sex, prosthetic conduit, and need of distal thrombectomy were independently associated with worse patency rates. Poor intra-operative runoff was correlated with higher amputation rates. CONCLUSION: Among those undergoing infrainguinal bypass, patients who present with acute limb ischaemia constitute a subset showing higher early rates of amputation and death. In this subset of patients, worse outcomes may be expected for women, prosthetic conduits, need for distal thrombectomy, and patients with poor intra-operative runoff.

[Anesthesia in the surgical repair of thoracoabdominal aneurysms: 17 years experience]. / Anestesia en la cirugía del aneurisma toracoabdominal: 17 años de experiencia.

OBJECTIVE: To review the results after thoracoabdominal aortic aneurysms repair performed by the same team of surgeons and anesthesiologists over a 17 year period. MATERIAL AND METHODS: A prospective and observational study carried out on 65 patients (97% male) who were operated on from 1995 until July 2011 for thoracoabdominal aneurysm, Crawford type i 5 (7.6%), ii 22 (33.8%), iii 17 (26.11%) and iv 21 (32.31%). RESULTS: The 30-day mortality was 9.2% (6/65 patients), which was the same as the incidence of paraplegia. This complication only occurred in the Crawford types ii and iii aneurysms. Paraplegia was present in 4.5% (1/22) of patients in whom "left bypass" was used, compared with 29% (5/17) in which the intervention was performed by cross-clamping without distal aortic perfusion (P=.068). The most frequent complication was respiratory, with prolonged mechanical ventilation (>48h) in 20% (13 patients) of cases. The mean hospital stay was 28 days (7-92). DISCUSSION: The average mortality in referral centers is 9.7%, ranging between 5% and 16%. In other centres the mortality at 30 days is between 19% and 31% per year. Our group had a 9.2% of mortality rate at 30 days. The incidence of paraplegia in hospitals with greater experience ranges between 2.7% and 16%. Nowadays, an incidence of less than 10% is accepted as good. We had 9.2%. The use of CSF drainage in these patients, as well as the use of left bypass and moderate hypothermia in the Crawford types i, ii and iii appear to be acceptable for prophylaxis of paraplegia.

A first evaluation of the usefulness of Kudzu starch in cultural heritage restoration.

In recent times, the use of natural and harmless products for the environment and restorer is taking place in the field of Cultural Heritage restoration. In this sense, wheat, rice and corn starches as adhesives, have suitable characteristics without toxicity risks. A new starch in this field, is the Kudzu, an almost pure compound (99.5% starch) that is processed by a natural way from a plant called Pueraria lobata. This is a preliminary study of the potential use of Kudzu starch for the restoration of Cultural Heritage, focusing, firstly, in its capacity as adhesive through a comparative evaluation with common starches. The accelerated aging process carried out proved that Kudzu ensures optimal chromatic behaviour. On the other hand, the main problem in starch paste is the biological colonization. The daidzein, a natural antimicrobial compound implicit in Kudzu starch, confirmed the resistance to microorganism in this preliminary approach. The evaluation of the adhesive capacity, and the reversibility of the starches, suggest that Kudzu starch is a valid adhesive in the field of paper restoration. Thus, the potential of this starch in the conservation of Cultural Heritage is evidenced and its use as cleaner, resistance to biological colonization and consolidant is promising.

Characterization of a monoclonal antibody directed against Salmonella enterica serovar Typhimurium and serovar [4,5,12:i:-].

Flagellar extracts of Salmonella enterica serovars expressing phase 2 H1 antigenic complex (H:1,2, H:1,5, H:1,6, and H:1,7) and a mutant flagellin obtained by site-directed mutagenesis of the fljB gene from serovar Typhimurium at codon 218, transforming threonine to alanine, expressed in Escherichia coli (fljB218(A)) were used to analyze the H1 antigenic complex. Cross-reactions were detected by Western blotting and dot blotting using commercial polyclonal antibodies against the different wild-type extracts and mutant FljB218(A). Therefore, we produced a monoclonal antibody (MAb), 23D4, isotyped as immunoglobulin M, against H:1,2 S. enterica serovar Typhimurium flagellin. The mutant flagellin was not recognized by this MAb. When a large number of phase 1 and phase 2 flagellin antigens of different serovars were used to characterize the 23D4 MAb, only extracts of serovars Typhimurium and [4,5,12:i:-] reacted. The protein composition of phase 1 and phase 2 extracts and highly purified H:1,2 flagellin from serovar Typhimurium strain LT2 and extract of strain 286 (serovar [4,5,12:i:-]), which reacted with the MAb, was studied. Phase 2 flagellin (FljB(H:1,2)) was detected in phase 1 and phase 2 flagellar heat extracts of serovar Typhimurium and was the single protein identified in all spots of purified H:1,2 flagellin. FliC, FlgK, and other proteins were detected in some immunoreactive spots and in the flagellar extract of serovar [4,5,12:i:-]. Immunoelectron microscopy of complete bacteria with 23D4 showed MAb attachment at the base of flagella, although the MAb failed to recognize the filament of flagella. Nevertheless, the results obtained by the other immunological tests (enzyme-linked immunosorbent assay, Western blotting, and dot blotting) indicate a reaction against flagellins. The epitopes could also be shared by other proteins on spots where FljB is not present, such as aminopeptidase B, isocitrate lyase, InvE, EF-TuA, enolase, DnaK, and others. In conclusion, MAb 23D4 can be useful for detection and diagnostic purposes of S. enterica serovar Typhimurium and serovar [4,5,12:i:-] and could be also helpful for epitope characterization of flagellum-associated antigens.

[Contralateral occlusion: does it affect the therapeutic management of carotid stenosis?]. / Oclusion contralateral: afecta al tratamiento de la estenosis carotidea?

INTRODUCTION: Because the contralateral carotid artery occlusion has been considered a high risk factor for ipsilateral carotid endarterectomy, some authors indicate a stenting procedure in these cases. AIM: To evaluate whether there are different preoperative factors in patients with contralateral carotid occlusion and if the latter is associated to worse perioperative outcome. PATIENTS AND METHODS: We analysed 476 carotid endarterectomies performed in 1994-2004. 57 cases (12.0%) had contralateral carotid occlusion. We analysed the results in patients with contralateral carotid occlusion and we compared them with the patients with contralateral patency. Prospective cohort study. RESULTS: There were no differences in preoperative risk factors or comorbidity. Contralateral occlusion was associated to preoperative symptoms: 75.4-58.9%; OR: 1.2 (1.1-1.5); p = 0.02. The patients operated on under local anaesthesia who had contralateral occlusion and previous stroke, had a higher rate of cerebral ischaemia during carotid cross-clamping: 66.7-11.3%; RR: 5.8 (3.1-10.9); p = 0.002. Morbimortality was 2.3% in the global series and 0.0% in patients with contralateral carotid occlusion. Over 80-year-old patients with contralateral carotid occlusion had a higher global rate of cardiac complications: 28.6-0.0% (p = 0.01). CONCLUSIONS: In our series, patients with contralateral carotid occlusion do not have a higher risk preoperative profile o higher perioperative morbimortality. Contralateral carotid occlusion does not justify, by itself, the endovascular treatment of an ipsilateral carotid stenosis.

[Immediate and long-term outcomes of carotid endarterectomies in eighty-year-olds]. / Resultados inmediatos y a largo plazo de la endarterectomía carotídea en octogenarios.

AIMS: To compare the immediate results obtained after performing a carotid endarterectomy (cEDA) in patients in their eighties with those of younger patients, and to determine survival and stroke-free times following carotid surgery in the two groups. PATIENTS AND METHODS: Retrospective data was collected regarding a series of 319 cases of cEDA (302 patients) performed between January 1998 and December 2004. Group 1: patients aged 80 or above. Group 2: the rest of the series. Sample follow-up: clinical and by means of carotid duplex. RESULTS: Mean age of the sample: 70.7 years (41-86). Group 1 was made up of 30 patients (9.4% of the series). Mortality rate: group 1, 3.3%; group 2, 1%; p=0.32. Major stroke-death incidence: group 1, 6.7%; group 2, 1.4%; p= 0.1. Median follow-up time: 36 months (1-87). Total mortality of the series throughout follow-up: 36 patients (12.6%). Mortality rate: group 1, 25%; group 2, 11.3%; p=0.04; relative risk: 2.6 (1.02-6.7). Stroke: group 1, 14.3%; group 2, 2.3%; p=0.01; relative risk: 7 (1.8-26.4). At five years, 96.7% were free of strokes (group 1: 84%; group 2, 97.7%; p=0.0001). At five years, 82.4% survived free of strokes (group 1: 61%; group 2, 84.4%; p=0.004). CONCLUSIONS: The risk of perioperative complications is higher in patients in their eighties than in younger patients, although the increase is not statistically significant. Even though the risk of a stroke during follow-up was higher in the eighty-year-olds, 84% of the subjects in this group remained stroke-free at five years. The high stroke-free survival rate in the medium to long term means that cEDA can be especially beneficial for patients in their eighties.

Oligo p-Phenylenevinylene Derivatives as Electron Transfer Matrices for UV-MALDI.

Phenylenevinylene oligomers (PVs) have outstanding photophysical characteristics for applications in the growing field of organic electronics. Yet, PVs are also versatile molecules, the optical and physicochemical properties of which can be tuned by manipulation of their structure. We report the synthesis, photophysical, and MS characterization of eight PV derivatives with potential value as electron transfer (ET) matrices for UV-MALDI. UV-vis analysis show the presence of strong characteristic absorption bands in the UV region and molar absorptivities at 355 nm similar or higher than those of traditional proton (CHCA) and ET (DCTB) MALDI matrices. Most of the PVs exhibit non-radiative quantum yields (φ) above 0.5, indicating favorable thermal decay. Ionization potential values (IP) for PVs, calculated by the Electron Propagator Theory (EPT), range from 6.88 to 7.96 eV, making these oligomers good candidates as matrices for ET ionization. LDI analysis of PVs shows only the presence of radical cations (M+.) in positive ion mode and absence of clusters, adducts, or protonated species; in addition, M+. threshold energies for PVs are lower than for DCTB. We also tested the performance of four selected PVs as ET MALDI matrices for analytes ranging from porphyrins and phthalocyanines to polyaromatic compounds. Two of the four PVs show S/N enhancement of 1961% to 304% in comparison to LDI, and laser energy thresholds from 0.17 µJ to 0.47 µJ compared to 0.58 µJ for DCTB. The use of PV matrices also results in lower LODs (low fmol range) whereas LDI LODs range from pmol to nmol. Graphical Abstract ᅟ.

Diadenosine polyphosphate receptors. from rat and guinea-pig brain to human nervous system.

Diadenosine polyphosphates are a family of naturally occurring nucleotidic compounds present in secretory vesicles together with other chemical messengers. The exocytotic release of these compounds permits them to stimulate receptors termed "purinoceptors" or "ATP receptors." Purinoceptors for nucleotides are named P2 in contrast with those sensitive to nucleosides (P1). P2 receptors are further subdivided into metabotropic P2Y receptors, further divided into 5 subtypes, and ionotropic P2X receptors, with 7 different subtypes. Diadenosine polyphosphates can activate recombinant P2Y(1), P2Y(2), and P2Y(4) and recombinant homomeric P2X(1), P2X(2), P2X(3), P2X(4), and P2X(6). Heteromeric P2X receptors change their sensitivity to diadenosine polyphosphates when co-assembly between different subunits occurs. Diadenosine polyphosphates can activate specific receptors termed dinucleotide receptors or P4 receptors, which are insensitive to other nucleosides or nucleotides. The P4 receptor is a receptor-operated Ca(2)+ channel present in rat brain synaptic terminals, stimulated by diadenosine pentaphosphate and diadenosine tetraphosphate. This receptor is strongly modulated by protein kinases A and C and protein phosphatases. The dinucleotide receptor is present in different brain areas, such as midbrain (in rat and guinea-pig), cerebellum (in guinea-pig), and cortex (in human).

A 50-gene signature is a novel scoring system for tumor-infiltrating immune cells with strong correlation with clinical outcome of stage I/II non-small cell lung cancer.

PURPOSE: To identify a novel system for scoring intratumoral immune response that can improve prognosis and therapy decisions in early stage non-small cell lung cancer (NSCLC). METHODS/PATIENTS: Eighty-four completely resected stage I/II NSCLC without adjuvant therapy were classified by expression profiling using whole genome microarrays. An external cohort of 162 tumors was used to validate the results. Immune cells present in tumor microenvironment were evaluated semiquantitatively by CD20, CD79, CD3, CD8, CD4 and CD57 immunostaining. Univariate and multivariate analyses of variables associated with recurrence-free survival were performed. RESULTS: Initial molecular classification identified three clusters, one with significantly better RFS. A reduced two-subgroup classification and a 50-gene predictor were built and validated in an external dataset: high and low risk of recurrence patients (HR = 3.44; p = 0.001). Analysis of the predictor´s genes showed that the vast majority were related to a B/plasma cell immune response overexpressed in the low-risk subgroup. The predictor includes genes coding for unique B lineage-specific genes, functional elements or other genes that, although non-restricted to this lineage, have strong influence on B-cell homeostasis. Immunostains confirmed increased B-cells in the low-risk subgroup. Gene signature (p < 0.0001) and CD20 (p < 0.05) were predictors for RFS, while CD79 and K-RAS mutations showed a tendency. CONCLUSIONS: Favorable prognosis in completely resected NSCLC is determined by a B-cell-mediated immune response. It can be differently scored by a 50-gene expression profile or by CD20 immunostaining. That prognosis information not reflected by traditional classifications may become a new tool for determining individualized adjuvant therapies.

Immunohistochemical localization of the vasopressin V1b receptor in the rat brain and pituitary gland: anatomical support for its involvement in the central effects of vasopressin.

Biological effects of vasopressin (VP) are mediated by four different receptors, two of which (the V1a and the oxytocin receptors) have been well characterized in the rodent brain, suggesting that these are the main receptors responsible for the central effects of VP. However, transcripts of the V1b VP receptor (V1bR) have been detected throughout the rat brain by RT-PCR and in situ hybridization, indicating that the V1bR adds to the population of central VP receptors. Because there are no specific ligands for the V1bR, the receptor protein itself has been difficult to visualize. In the present study, the distribution of the V1bR protein was investigated in the rat forebrain, midbrain, hindbrain, and cerebellum by immunohistochemistry using an antiserum raised against a synthetic fragment of the carboxylterminal of the rat V1bR protein. Immunohistochemistry revealed the presence of the V1bR in pituitary corticotrophs as expected. In naive, untreated rats, fiber networks containing V1bR-immunoreactivity were mainly concentrated in the hypothalamus, amygdala, cerebellum, and particularly in those areas with a leaky blood brain barrier or close to the circumventricular organs (medial habenula, subfornical organ, organum vasculosum laminae terminalis, median eminence, and nuclei lining to the third and fourth ventricles). A strikingly dense network was present in the external zone of the median eminence. Colchicine treatment was required to reveal the localization of V1bR-immunoreactive cell bodies. V1bR-containing cell bodies and associated protrusions were mainly located in the hippocampus, caudate putamen, cortex, thalamus, olfactory bulb, and cerebellum. These results demonstrate the widespread distribution of the V1bR protein in the rat brain over multiple, functionally distinct neuronal systems. These data suggest that the V1bR mediates different physiological functions of VP in the brain.

Differential responses of phosphorylated mitogen-activated protein kinase and phosphorylated cyclic-AMP response element-binding protein immunoreactivity in the rat brain to sub-convulsive pentylenetetrazol.

The possible advantage of using multiple phospho-specific antibodies to study changes in brain activity was assessed. For this purpose, rats were injected intraperitoneally with either a control treatment or 15 mg/kg pentylenetetrazol. The sub-convulsive dose of pentylenetetrazol did not induce marked behavioural effects. Ten minutes after treatment, the rats were perfused and the brains were dissected. Adjacent brain sections were immunohistochemically stained for phosphorylated cyclic-AMP response element-binding protein and phosphorylated mitogen-activated protein kinase. Opposite effects of pentylenetetrazol treatment were observed on the immunoreactivity of these two antibodies within the hypothalamic paraventricular nucleus, the supraoptic nucleus and the arcuate nucleus. In these regions, pentylenetetrazol treatment increased phosphorylated mitogen-activated protein kinase immunoreactivity, but decreased phosphorylated cyclic-AMP response element-binding protein immunoreactivity. These findings show that changes in the activity of a brain nucleus can be accompanied by differential changes in the activity of two signal transduction pathways, which can be detected immunohistochemically. Therefore, the use of multiple phospho-specific antibodies may enhance our potential to monitor changes in brain activity.

Impairment of stress adaptive behaviours in rats by the CCKA receptor antagonist, devazepide.

1. Cholecystokinin (CCK) is released during stress both in limbic and hypothalamic areas suggesting that CCK could participate in modulating neuroendocrine as well as behavioural responses to stress. 2. In this study we have examined the effect of CCK receptor antagonists on the retention of the immobility response to a forced-swim stress in rats. In this test, rats are forced to swim during 15 min (conditioning period) and 24 h later, the duration of immobility is measured during a period of 5 min (re-test period). During the conditioning period rats display a period of vigorous activity, followed by progressive inactivity. During the re-test period rats remain 70-80% of the time in an immobile posture. 3. The CCKA receptor antagonist, devazepide (MK-329) but not the CCKB receptor antagonist, L-365,260, administered s.c. immediately before the conditioning period, decreased the duration of acquired immobility during the re-test period. The effect of devazepide was prevented by cholecystokinin octapeptide (CCK-8; 40 micrograms kg-1, s.c) as well as by the selective glucocorticosteroid GII receptor agonist, dexamethasone (30 micrograms kg-1, s.c.) 4. Neither corticosterone nor ACTH plasma levels measured both after the re-test period and after the conditioning period were modified by devazepide treatment. 5. The results suggest a role for CCK in the behavioural adaptation to stress and indicate a relationship between CCK systems and glucocorticoids in the neuronal mechanisms involved in the acquisition of adaptive behaviours to stress.

Biochemistry of vasopressin fragments.

Vasopressin (VP) undergoes a step-wise aminopeptidase conversion process in the brain, leading to accumulation of several metabolites. Some of these metabolites, in particular [pGlu4,Cyt6]VP 4-9 and 4-8, show behavioral effects comparable to VP, but are more potent and selective than VP. Most data favor the existence of a separate receptor for the VP metabolites distinct of the classical VP and oxytocin receptors, although its identity has remained obscure thus far. The characterization of this receptor is a major challenge to understand how the brain VP system generates and regulates divers central functions.

Prognostic value of the quantified expression of p185 in non-small cell lung cancer.

BACKGROUND: We sought to assess the relationship between tissue concentration of erb -b-2 or neu oncogene-encoded protein (p185(neu)) with overall survival in patients with non-small cell lung cancer. METHODS: Levels of protein p185(neu) were determined in 102 patients with the diagnosis of non-small cell lung cancer. Concentration of p185(neu) protein was determined by using enzyme immunoassay and evaluated by using several variables. The relative prognostic importance of this marker and its influence on other prognostic factors was evaluated by using the Cox regression model. RESULTS: The mean p185(neu) value in these samples was 250 +/- 200 U/mg (95% confidence interval, 210-290). This distinguished two groups within the tumoral population: those with less than 350 U/mg and those with 350 U/mg or greater (80th percentile). Multivariable analysis established an independent prognostic value for protein p185(neu). Patients with p185(neu) values of the 80th percentile or greater had a risk of death that was 2.11-fold (95% confidence interval, 1.10-4.05) that of patients with values of less than 350 U/mg (P =.03), and increases in the neu oncogene of 100 U/mg increased the probability of death by 17% (P =.02; 95% confidence interval, 1.04-1.31). CONCLUSION: This study shows that the p185(neu) expression is an objective and comparable variable for the assessment of phenotypic aggressivity in non-small cell lung cancer, and in the future, it could be included in daily clinical practice.

The CCKB receptor antagonist, L-365,260, elicits antidepressant-type effects in the forced-swim test in mice.

Selective CCKA and CCKB receptor agonists and antagonists were used to study the involvement of endogenous cholecystokinin in the behavioural changes that occur in mice in the forced-swimming test (Porsolt's test). The CCKB receptor antagonist, L-365,260 ((3R)-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4- benzodiazepin-3-yl)-3-methylphenylurea), but not the CCKA receptor antagonist, devazepide ((3S)-(-)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin -3-yl)- 1H-indole-2-carboxamide), elicited an antidepressant-type response (a decrease in the duration of immobility) that was suppressed by previous treatment with either CCK-8 (H-Asp-Tyr(OSO3H)-Met-Gly- Trp-Met-Asp-Phe-NH2) or the selective CCKB receptor agonist BC-264 (Boc-Tyr(SO3H)-gNle-mGly-Trp-N(Me)-Nle-Asp-Phe- NH2). The L-365,260 effect was also prevented by the dopamine receptor antagonist, SCH-23,390 (a dopamine D1-selective receptor antagonist: R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl- 2,3,4,5-tetrahydro-1H-3-benzazepine) and sulpiride (a dopamine D2-selective receptor antagonist: (-)-5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl) methyl]-2-metoxybenzamide). On the other hand, co-administration of subthreshold doses of L-365,260 and nomifensine (an atypical antidepressant that selectively blocks dopamine re-uptake mechanisms, 1,2,3,4-tetrahydro-2-methyl-4-phenyl-8-isoquinolinamine) led to a potent antidepressant-type response. These results indicate that blocking of CCKB receptors could result in an increase of extracellular dopamine contents in some brain areas involved in depression and suggest a potential use of CCKB receptor antagonists, alone or combined with antidepressants, in the treatment of depressive syndromes.

Antidepressant-like effects of CCK(B) receptor antagonists: involvement of the opioid system.

RB 101 (N-[(R,S)-2-benzyl-3-[(S)-2-amino-4-methylthiobutyldithio]-1-oxopr opyl]-L -phenylalaninebenzyl ester), a systemically active inhibitor of enkep halin catabolism, has been shown to elicit antidepressant-like effects in mice, both in the forced-swimming and in the conditioned suppression of the mobility tests. The same type of response has been also observed following administration of the cholecystokinin CCK(B) receptor antagonist L-365,260 ((3R)-(+)-N-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin -3-yl)-3 -methylphenylurea). In terestingly, the delta-opioid receptor antagonist naltrindole (17-cyclopropylmethyl-6,7-dehydro-4,5alpha-epoxy-3,14-dihydroxy-6, 7,2'-3'-indolomorphinan) blocks the effect of both RB 101 and L-365,260 in the conditioned suppression of the motility test. In this work we have investigated the involvement of the opioid system in the antidepressant response to the CCK(B) receptor antagonist L-365,260 in the forced-swimming test in mice. The effect of L-365,260 was decreased by the delta-opioid receptor antagonist naltrindole. Furthermore, the CCK(B) receptor agonist, BC 264 (Boc-Tyr(OSO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2), blocked the antidepressant-like effect of RB 101 while CCK-8 (H-Asp-Tyr(OSO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) enhanced the effect of this drug, probably through stimulation of central CCK(A) receptors, since the CCK(A) receptor antagonist devazepide ((3S)-(-)-(2,3-dihydro-1-methyl-2-oxo-5-phenyl-1H-1,4-benzodiazepin++ +-3-yl)-1H-indole-2 -carboxamide) abolished the CCK-8-induced potentiation of the RB 101 effect. In addition, RB 101 enhanced the effect of L-365,260. Such an effect was blocked by the delta-opioid receptor antagonist naltrindole. These data further support the involvement of opioid receptors in the antidepressant-type effect induced by CCK(B) receptor blockers and support the hypothesis of a regulatory role of CCK in the activity of the endogenous opioid system. As in other experimental paradigms, CCK(A) and CCK(B) receptor stimulation appears to have opposite effects in modulating opioidergic activity.

Resistance to candidiasis and macrophage activity in chitin-treated mice.

The effect of chitin, a polysaccharide of the cell wall of Candida albicans, on both the survival of C. albicans infected mice and the activity of the murine peritoneal macrophages has been studied. Pretreatment of mice with 30 mg kg(-1) C. albicans chitin enhanced the survival of the infected animals. The protective effect was concomitant with an enhancement of both phagocytic and candidacidal activities of the peritoneal macrophages. Chitin by itself did not induce the nitric oxide (NO) synthase in the macrophages, which remained at a level similar to that shown by the macrophages from untreated animals. The administration of 10 mg kg(-1) C. albicans chitin diminished the long term survival of the infected animals. This effect was coincident with a lower candidacidal activity and NO production by the macrophages of the chitin treated and infected animals, compared to the untreated infected animals.

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence.

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.

Presence of different ATP receptors on rat midbrain single synaptic terminals. Involvement of the P2X(3) subunits.

Adenosine 5'-triphosphate (ATP) stimulates a [Ca(2+)](i) increase via specific ionotropic receptors, termed P2X receptors, in rat midbrain presynaptic terminals. A microfluorimetric technique enabled study of the [Ca(2+)](i) increase in isolated single synaptic terminals, showing that 33.4+/-2.5% of them responded to ATP. Immunological studies carried out, after functional studies, with specific anti-P2X receptor subunit antibodies showed only positive labelling with anti-P2X(3) antibodies in 23.5+/-1.7% of the terminals. All positively P2X(3) labelled synaptic terminals responded to ATP. Nevertheless, not all of them responded to alpha,beta-meATP, these representing 6.7+/-1.5% of the total. In addition, 9.8+/-2.3% of the terminals responded to ATP but exhibit negative P2X(3)-labelling. These results demonstrate the existence of a heterogeneous population of ionotropic ATP receptors at the presynaptic level.