Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Application de la modélisation moléculaire à de nouvelles approches thérapeutiques du cancer [Application of molecular modeling to new therapeutic cancer approaches].

Recent progress in the experimental determination of protein structures allow to understand, at a very detailed level, the molecular recognition mechanisms that are at the basis of the living matter. This level of understanding makes it possible to design rational therapeutic approaches, in which effectors molecules are adapted or created de novo to perform a given function. An example of such an approach is drug design, were small inhibitory molecules are designed using in silico simulations and tested in vitro. In this article, we present a similar approach to rationally optimize the sequence of killer T lymphocytes receptors to make them more efficient against melanoma cells. The architecture of this translational research project is presented together with its implications both at the level of basic research as well as in the clinics.

Age differences in the EEG N2 and P3 components during an interference task

The age-related increase in interference susceptibility has been well documented and largely attributed to a deficit in inhibition. In the present study, event-related potentials were used to investigate EEG correlates of inhibitory processing in an interference "Arrow" task. A specific interest was addressed to theN2 and P3 components that respectively refers to conflict monitoring and to efficiency of inhibition processes (Anguera et al,. 2011). Younger (N=10, Mage=24.6) and older (N=10, Mage=65.5) participants were invited to perform a task consisting in deciding, as fast and accurately as possible, whether an arrow presented on a computer screen points to the left or the right, irrespective of its position on the screen (left, middle or right). Responses were provided by key-presses using the left and right indexes. Three conditions were considered: congruent (arrow pointing to the same direction as that of the side of the screen on which it appears), incongruent (arrow pointing to the opposite direction), and neutral (arrow presented at the center of the screen). A total of 56 trials per conditions were performed. Behaviorally, the results showed that in the incongruent condition the percent of correct responses significantly decreased in both groups. After adjustment with simple RT (additional control task), the increased RTs obtained in the old group were significantly more pronounced in the incongruent condition. With respect to electrophysiological data, results showed that frontal site (Fz), the N2 amplitude was significantly larger for the younger as compared to the older (- 2.55 μV vs. -0.62 μV respectively) whatever the condition. At central site (Cz), the P3 amplitude significantly decreased in the older compared to the younger in the incongruent condition only. Our findings suggest that the increased RTs observed in older participants during the incongruent condition is more specifically linked to late cognitive resources involved in inhibiting prepotent response tendencies rather than associated with earlier stages of treatment dedicated to conflict monitoring.

Cytotoxic and Antiviral Activities of Colombian Medicinal Plant Extracts of the Euphorbia genus

Forty-seven plant extracts of 10 species of the genus Euphorbia (Euphorbiaceae) used by Colombian traditional healers for the treatment of ulcers, cancers, tumors, warts, and other diseases, were tested in vitro for their potential antitumour (antiproliferative and cytotoxic) and antiherpetic activity. To evaluate the capacity of the extracts to inhibit the lytic activity of herpes simplex virus type 2 (HSV-2) and the reduction of viability of infected or uninfected cell cultures, the end-point titration technique (EPTT) and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric assay were used, respectively. The therapeutic index of the positive extracts for the antiviral activity was determined by calculating the ratio CC50 (50% cytotoxic concentration) over IC50 (50% inhibitory concentration of the viral effect). Five of the 47 extracts (11%) representing 3 out of 10 Euphorbia species (30%) exhibited antiherpetic action; the highest activity was found in the leaf/stem water-methanol extracts from E. cotinifolia and E. tirucalli. The therapeutic indexes of these two plant species were > 7.1; these extracts exhibited no cytotoxicity. Six extracts (13%) representing 4 plant species (40%) showed cytotoxic activity. The highest cytotoxicity was found in the dichloromethane extract obtained from E. cotinifolia leaves and the CC50 values for the most susceptible cell lines, HEp-2 and CHO, were 35.1 and 18.1 µg/ml, respectively.

Funcions de les molècules associades a la mielina i la proteïna priònica cel•lular (PrPc) en neurodegeneració (Malaltia d’Alzheimer) i neuroinflamació (Esclerosi Múltiple)

Les proteïnes associades a la mielina (MAIS), Nogo-A, MAG i OMgp, són molècules que presenten una capacitat inhibitòria molt important per el recreixement axonal i la neuroreparació després de lesió. No obstant des de fa anys les seves funcions han estat ampliades i s’han involucrat en diferents processos degeneratius del sistema nerviós o en processos neuroinflamatoris del sistema nerviós central i el perifèric com ara l'Escleresi Múltiple (MS). La base neurobiològica d’indicadors moleculars que són responsables del dany axonal en MS segueixen sense estar plenament descrits. Recentment s’ha publicat que el mecanisme de senyalització Nogo-A pot regir els primers canvis de la desmielinització immunomediada del sistema nerviós central en el model animal de MS, l’encefalomielitis autoimmune experimental (EAE). De la mateixa forma la proteïna priònica cel•lular és una proteïna que s’ha associat majoritàriament a malalties espongiformes, però que recentment s’ha vinculat (no sense controvèrsia) amb la seva possible relació amb la Malaltia d'Alzheimer (AD), ja que seria capaç de reclutar els oligòmers d’Aβ (ADDLs), els quals correlacionen millor amb el grau de demència, i amb els que interacciona directament, actuant així com un possible mediador de la fosforilació de tau en la malaltia. No obstant, les funcions de les MAIS i de la PrPc en aquests models de la malaltia no estan clarament definits i, per altra banda, es desconeixen els mecanismes de senyalització implicats, no descartant de forma clara el component neural i l’immune.

When solving 22-7 is much more difficult than 99-12.

We describe the case of a 69-year-old professor of mathematics (GV) who was examined 2 years after left-hemispheric capsular-thalamic haemorrhage. GV showed disproportionate impairment in subtractions requiring borrowing (22 - 7). For large subtraction problems without borrowing (99 - 12) performance was almost flawless. Subtractions with borrowing mostly relied on inadequate attempts to invert subtractions into the corresponding additions (22 - 7 = x as 7 + x = 22). The hypothesis is advanced that difficulty in the inhibitory components of attention tasks (Stroop test, go-no-go task) might be the responsible factor of his calculation impairment. A deficit in subtractions with borrowing might be related to left-hemispheric damage involving thalamo-cortical connections.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Cre recombinase-mediated inactivation of H-2Dd transgene expression: evidence for partial missing self-recognition by Ly49A NK cells.

We have established H-2D(d)-transgenic (Tg) mice, in which H-2D(d) expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2D(d) Tg in H-2(b) mice and acquired reactivity to cells lacking H-2D(d), both in vivo and in vitro. H-2D(d)-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2D(d) expression. That abrogated NK cell reactivity to cells lacking D(d). In D(d) single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking D(d), because the inhibitory Ly49A receptor is no longer engaged by its D(d) ligand. In contrast, Ly49A+ NK cells from D(d) x MxCre double Tg mice were unable to react to D(d)-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated D(d) expression thus impacts specifically missing D(d) but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of D(d) from some host cells results in the acquisition of only partial missing self-reactivity.

Education of Murine NK Cells Requires Both cis and trans Recognition of MHC Class I Molecules.

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I.

Antibacterial xanthones from Kielmeyera variabilis mart. (Clusiaceae)

The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w). The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 µg/ml. When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 µg/ml against B. subtilis. Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains. None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa. Viable cells of S. aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture. It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved

cAMP prevents the glucose-mediated stimulation of GLUT2 gene transcription in hepatocytes.

Glucose homoeostasis necessitates the presence in the liver of the high Km glucose transporter GLUT2. In hepatocytes, we and others have demonstrated that glucose stimulates GLUT2 gene expression in vivo and in vitro. This effect is transcriptionally regulated and requires glucose metabolism within the hepatocytes. In this report, we further characterized the cis-elements of the murine GLUT2 promoter, which confers glucose responsiveness on a reporter gene coding the chloramphenicol acetyl transferase (CAT) gene. 5'-Deletions of the murine GLUT2 promoter linked to the CAT reporter gene were transfected into a GLUT2 expressing hepatoma cell line (mhAT3F) and into primary cultured rat hepatocytes, and subsequently incubated at low and high glucose concentrations. Glucose stimulates gene transcription in a manner similar to that observed for the endogenous GLUT2 mRNA in both cell types; the -1308 to -212 bp region of the promoter contains the glucose-responsive elements. Furthermore, the -1308 to -338 bp region of the promoter contains repressor elements when tested in an heterologous thymidine kinase promoter. The glucose-induced GLUT2 mRNA accumulation was decreased by dibutyryl-cAMP both in mhAT3F cells and in primary hepatocytes. A putative cAMP-responsive element (CRE) is localized at the -1074/-1068 bp region of the promoter. The inhibitory effect of cAMP on GLUT2 gene expression was observed in hepatocytes transfected with constructs containing this CRE (-1308/+49 bp fragment), as well as with constructs not containing the consensus CRE (-312/+49 bp fragment). This suggests that the inhibitory effect of cAMP is not mediated by the putative binding site located in the repressor fragment of the GLUT2 promoter. Taken together, these data demonstrate that the elements conferring glucose and cAMP responsiveness on the GLUT2 gene are located within the -312/+49 region of the promoter.

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiânia, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.

Antimicrobial activity in vitro of plumbagin isolated from Plumbago species

Plumbagin is a naturally occurring naphthoquinone isolated from roots of Plumbago scandens. The plant was collected at the Campus of Fundação Oswaldo Cruz, Rio de Janeiro, Brazil. P. scandens is used as a traditional medicine for the treatment of several diseases. The antimicrobial activity of plumbagin was evaluated using the macrodilution method. The compound exhibited relatively specific activity against bacteria and yeast. The minimum inhibitory concentration test showed the growth inhibiton of Staphylococcus aureus at a concentration of 1.56 µg/ml and of Candida albicans at a concentration of 0.78 µg/ml. These results suggest the naphthoquinone plumbagin as a promising antimicrobial agent.

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extractdisplayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 µg/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 µg/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 µg/ml) and B. subtilis (MIC at 3.9 and 7.8 µg/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 µg/ml and 3.12 µg/ml, respectively. Both compounds presented MIC of 3.12 µg/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 µg/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.

Discovery of amino acid variants in the human glucose-dependent insulinotropic polypeptide (GIP) receptor: the impact on the pancreatic beta cell responses and functional expression studies in Chinese hamster fibroblast cells.

The two incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), are insulinotropic factors released from the small intestine to the blood stream in response to oral glucose ingestion. The insulinotropic effect of GLP-1 is maintained in patients with Type II (non-insulin-dependent) diabetes mellitus, whereas, for unknown reasons, the effect of GIP is diminished or lacking. We defined the exon-intron boundaries of the human GIP receptor, made a mutational analysis of the gene and identified two amino acid substitutions, A207 V and E354Q. In an association study of 227 Caucasian Type II diabetic patients and 224 matched glucose tolerant control subjects, the allelic frequency of the A207 V polymorphism was 1.1% in Type II diabetic patients and 0.7% in control subjects (p = 0.48), whereas the allelic frequency of the codon 354 polymorphism was 24.9% in Type II diabetic patients versus 23.2% in control subjects. Interestingly, the glucose tolerant subjects (6% of the population) who were homozygous for the codon 354 variant had on average a 14% decrease in fasting serum C-peptide concentration (p = 0.01) and an 11% decrease in the same variable 30 min after an oral glucose load (p = 0.03) compared with subjects with the wild-type receptor. Investigation of the function of the two GIP receptor variants in Chinese hamster fibroblasts showed, however, that the GIP-induced cAMP formation and the binding of GIP to cells expressing the variant receptors were not different from the findings in cells expressing the wildtype GIP receptor. In conclusion, amino acid variants in the GIP receptor are not associated with random Type II diabetes in patients of Danish Caucasian origin or with altered GIP binding and GIP-induced cAMP production when stably transfected in Chinese hamster fibroblasts. The finding of an association between homozygosity for the codon 354 variant and reduced fasting and post oral glucose tolerance test (OGTT) serum C-peptide concentrations, however, calls for further investigations and could suggest that GIP even in the fasting state regulates the beta-cell secretory response.