A comparative study of rabies virus isolates from hematophagous bats in Brazil

The Brazilian chiropteran fauna consists of 167 species; of which, three are hematophagous: the common vampire bat (Desmodus rotundus), the white-winged vampire bat (Diaemus youngi), and the hairy-legged vampire bat (Diphylla ecaudata). The aim of this study was to describe the isolation of Rabies virus from common and hairy-legged vampire bats and to report the first comparative antigenic and genetic studies of isolates from these bats. Antigenic and genetic typing of both isolates identified them as antigenic variant 3 (AgV3), the variant frequently isolated from common vampire bats. Phylogenetic analysis showed 99.3% identity between the isolates. This is the first time since 1934 that Rabies virus has been isolated from hairy-legged vampire bats in Brazil. Our analysis provides evidence that the existence of rabies-positive isolates from hairy-legged vampire bats may be the result of an interspecific rabies transmission event from common vampire bats and suggests that roost cohabitation may occur.     

Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

Introduction

More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.

Methods

Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.

Results

Circulating CD14⁺ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.

Conclusions

Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.     

Response of the ant community to attributes of fragments and vegetation in a northeastern Atlantic Rain Forest area, Brazil

The objective of this study was to determine the effects of forest fragmentation on ant richness in a landscape of Atlantic Forest in Northeast Brazil. More specifically, the ant richness was related to the attributes of fragments (area and distance from the fragment central point to the edge), landscape (forest cover surrounding the fragments), and tree community (plant density, richness, and percentage of shade tolerant species). The surveys were carried out in 19 fragments located in Alagoas State from October 2007 to March 2008. Samples were collected through a 300 m transect established in the center of each fragment, where 30 1-m2 leaf litter samples were collected at 10 m intervals. A total of 146 ant species was collected, which belonged to 42 genera, 24 tribes and nine subfamilies. The attributes of fragments and landscape did not influence ant richness. On the other hand, tree density explained ca. 23% of ant richness. In relation to functional groups, both density and richness of trees explained the richness of general myrmicines (the whole model explained ca. 42% of the variation in this group) and percentage of shade tolerant trees explained the richness of specialist predator ants (30% for the whole model). These results indicate that ant fauna is more influenced by vegetation integrity than by fragment size, distance to edge or forest cover surrounding fragments.     

The interaction of Phe472 with a fluorescent inhibitor bound to the complex of myosin subfragment-1 with nucleotide

The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA) acts as a fluorescent inhibitor for the ATPases of skeletal [Hiratsuka (1994) J. Biol. Chem. 269, 27251-27257] and Dictyostelium discoideum [Bobkov et al. (1997) J. Muscle Res. Cell Motil. 18, 563-571] myosins. The former paper suggested that, upon addition of excess nucleotides to the binary complex of subfragment-1 from skeletal myosin (S1) with PPBA, a stable ternary complex of S1 with PPBA and nucleotide is formed. Useful fluorescence properties of PPBA enable us to distinguish the conformation of the myosin ATPase at the ATP state from that at the ADP state. In the present paper, to determine the PPBA-binding site in the complexes, enzymatic and fluorescence properties of the S1.PPBA.nucleotide complexes were investigated. Upon formation of the ternary complex with ATP, a new peak appeared at 398 nm in the PPBA fluorescence spectrum. Experiments using model compounds of aromatic amino acid suggested that this fluorescence peak at 398 nm is originated from PPBA interacting with Phe residue(s). Taking into account differences in fluorescence spectra between complexes of S1 and those of subfragment-1 from D. discoideum myosin (S1dC), in the ternary complex of S1 formed with ATP, PPBA was suggested to interact with Phe residue(s) that is absent in S1dC. Docking simulation of PPBA on the S1.nucleotide complex revealed that Phe472 interacts with PPBA. Binding sites of PPBA and blebbistatin, an inhibitor showing high affinity and selectivity toward myosin II [Kovács et al. (2004) J. Biol. Chem. 279, 35557-35563], seem to overlap at least partly.     

Selection on the human immunodeficiency virus type 1 proteome following primary infection

Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at < or =17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.     

Automated systems in the identification and determination of methicillin resistance among coagulase negative staphylococci

Coagulase-negative staphylococci (CoNS) are an important cause of nosocomial bacteremia, specially in patients with indwelling devices or those submitted to invasive medical procedures. The identification of species and the accurate and rapid detection of methicillin resistance are directly dependent on the quality of the identification and susceptibility tests used, either manual or automated. The objective of this study was to evaluate the accuracy of two automated systems--MicroScan and Vitek--in the identification of CoNS species and determination of susceptibility to methicillin, considering as gold standard the biochemical tests and the characterization of the mecA gene by polymerase chain reaction, respectively. MicroScan presented better results in the identification of CoNS species (accuracy of 96.8 vs 78.8%, respectively); isolates from the following species had no precise identification: Staphylococcus haemolyticus, S. simulans, and S. capitis. Both systems were similar in the characterization of methicillin resistance. The higher discrepancies for gene mec detection were observed among species other than S. epidermidis (S. hominis, S. saprophyticus, S. sciuri, S. haemolyticus, S. warneri, S. cohnii), and those with borderline MICs.     

A lentiviral vector with expression controlled by E2F-1: a potential tool for the study and treatment of proliferative diseases

We have constructed a lentiviral vector with expression limited to cells presenting active E2F-1 protein, a potential advantage for gene therapy of proliferative diseases. For the FE2FLW vector, the promoter region of the human E2F-1 gene was utilized to drive expression of luciferase cDNA, included as a reporter of viral expression. Primary, immortalized, and transformed cells were transduced with the FE2FLW vector and cell cycle alterations were induced with serum starvation/replacement, contact inhibition or drug treatment, revealing cell cycle-dependent changes in reporter activity. Forced E2F-1 expression, but not E2F-2 or E2F-3, increased reporter activity, indicating a major role for this factor in controlling expression from the FE2FLW virus. We show the utility of this vector as a reporter of E2F-1 and proliferation-dependent cellular alterations upon cytotoxic/cytostatic treatment, such as the introduction of tumor suppressor genes. We propose that the FE2FLW vector may be a starting point for the development of gene therapy strategies for proliferative diseases, such as cancer or restinosis.     

Angiotensin II disrupts inhibitory avoidance memory retrieval

The brain renin-angiotensin system (RAS) is involved in learning and memory, but the actual role of angiotensin II (A(II)) and its metabolites in this process has been difficult to comprehend. This has been so mainly due to procedural issues, especially the use of multi-trial learning paradigms and the utilization of pre-training intracerebroventricular infusion of RAS-acting compounds. Here, we specifically analyzed the action of A(II) in aversive memory retrieval using a hippocampal-dependent, one-trial, step-down inhibitory avoidance task (IA) in combination with stereotaxically localized intrahippocampal infusion of drugs. Rats bilaterally implanted with infusion cannulae aimed to the CA1 region of the dorsal hippocampus were trained in IA and tested for memory retention 24 h later. We found that when given into CA1 15 min before IA memory retention test, A(II), but not angiotensin IV or angiotensin(1-7) induced a dose-dependent and reversible amnesia without altering locomotor activity, exploratory behavior or anxiety state. The effect of A(II) was blocked in a dose-dependent manner by the A(II)-type 2 receptor (AT(2)) antagonist PD123319 but not by the A(II)-type 1 receptor (AT(1)) antagonist losartan. By themselves, neither PD123319 nor losartan had any effect on memory expression. Our data indicate that intra-CA1 A(II) hinders retrieval of avoidance memory through a process that involves activation of AT(2) receptors.     

Interleukin-1 mediates endothelin-1-induced fever and prostaglandin production in the preoptic area of rats

The intracerebroventricular injection of endothelin-1 (ET-1) induces fever and increases PG levels in the cerebrospinal fluid of rats. Likewise, the injection of IL-1 into the preoptic area (POA) of the rat hypothalamus causes both fever and increased PG production. In this study, we conducted in vivo and in vitro experiments in the rat to investigate 1) the hypothalamic region involved in ET-1-induced fever and PG biosynthesis and 2) whether hypothalamic IL-1 plays a role as a mediator of the above ET-1 activities. One hundred femtomoles of ET-1 increased body temperature when injected in the POA of conscious Wistar rats; this effect was significantly counteracted by the coinjection of 600 pmol IL-1 receptor antagonist (IL-1ra). In experiments on rat hypothalamic explants, 100 nM ET-1 caused a significant increase in PGE2 production and release from the whole hypothalamus and from the isolated POA, but not from the retrochiasmatic region, in 1-h incubations. Six nanomoles of IL-1ra or 10 nM of a cell-permeable interleukin-1 converting enzyme inhibitor completely counteracted the effect of ET-1 on PGE2 release from the POA. One hundred nanomoles ET-1 also caused a significant increase in IL-1beta immunoreactivity released into the bath solution of hypothalamic explants after 1 h of incubation, although during such time ET-1 failed to modify the gene expression of IL-1beta and other pyrogenic cytokines within the hypothalamus. In conclusion, our results show that ET-1 increases IL-1 production in the POA, and this effect appears to be correlated to ET-1-induced fever in vivo, as well as to PG production in vitro.     

Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells

Oxidized l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10-50 microg/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. O2-* production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of O2-* by xanthine oxidase-lumazine and H2O2 by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species.