Ma Isabel Salazar

Comparison of antioxidant activity of hydroethanolic fresh and aged garlic extracts and their effects on cerebral ischemia

Antioxidant properties and protective effect of aged garlic extract (AGE) and of 20% hydroethanolic fresh extracts from garlic clove (GCE) and skin (GSE) on cerebral ischemia were evaluated by administering extracts at the beginning of reperfusion in a rat model of stroke. All three extracts scavenged superoxide anion, peroxynitrite anion, and peroxyl radicals, but with different efficiencies; furthermore, GCE and GSE scavenged hydroxyl radicals and GSE scavenged singlet oxygen. These extracts significantly prevented reduction of neuronal nuclear antigen in the infarcted area, although no improvement in neurological function was observed. Importantly, GCE and GSE contained S-allylcystein, a compound associated with AGEs neuroprotective effect against damage induced by cerebral ischemia. Extracts decreased mRNA expression of NR1- and NR2B-NMDA-receptor subunits and prevented ischemia-induced reduction in mitochondrial potential and in ATP synthesis. These results indicate that antioxidants present in garlic extracts may regulate ROS concentrations during ischemia, favour pro-survival pathways, and attenuate mitochondrial dysfunction.

Vectorial Capacity of Aedes aegypti for Dengue Virus Type 2 Is Reduced with Co-infection of Metarhizium anisopliae

Background Aedes aegypti, is the major dengue vector and a worldwide public health threat combated basically by chemical insecticides. In this study, the vectorial competence of Ae. aegypti co-infected with a mildly virulent Metarhizium anisopliae and fed with blood infected with the DENV-2 virus, was examined. Methodology/Principal Findings The study encompassed three bioassays (B). In B1 the median lethal time (LT50) of Ae. aegypti exposed to M. anisopliae was determined in four treatments: co-infected (CI), single-fungus infection (SF), single-virus infection (SV) and control (C). In B2, the mortality and viral infection rate in midgut and in head were registered in fifty females of CI and in SV. In B3, the same treatments as in B1 but with females separated individually were tested to evaluate the effect on fecundity and gonotrophic cycle length. Survival in CI and SF females was 70% shorter than the one of those in SV and control. Overall viral infection rate in CI and SV were 76 and 84% but the mortality at day six post-infection was 78% (54% infected) and 6% respectively. Survivors with virus in head at day seven post-infection were 12 and 64% in both CI and SV mosquitoes. Fecundity and gonotrophic cycle length were reduced in 52 and 40% in CI compared to the ones in control. Conclusion/Significance Fungus-induced mortality for the CI group was 78%. Of the survivors, 12% (6/50) could potentially transmit DENV-2, as opposed to 64% (32/50) of the SV group, meaning a 5-fold reduction in the number of infective mosquitoes. This is the first report on a fungus that reduces the vectorial capacity of Ae. aegypti infected with the DENV-2 virus.

Large-Scale Genomic Analysis of Codon Usage in Dengue Virus and Evaluation of Its Phylogenetic Dependence

The increasing number of dengue virus (DENV) genome sequences available allows identifying the contributing factors to DENV evolution. In the present study, the codon usage in serotypes 1–4 (DENV1–4) has been explored for 3047 sequenced genomes using different statistics methods. The correlation analysis of total GC content (GC) with GC content at the three nucleotide positions of codons (GC1, GC2, and GC3) as well as the effective number of codons (ENC, ENCp) versus GC3 plots revealed mutational bias and purifying selection pressures as the major forces influencing the codon usage, but with distinct pressure on specific nucleotide position in the codon. The correspondence analysis (CA) and clustering analysis on relative synonymous codon usage (RSCU) within each serotype showed similar clustering patterns to the phylogenetic analysis of nucleotide sequences for DENV1–4. These clustering patterns are strongly related to the virus geographic origin. The phylogenetic dependence analysis also suggests that stabilizing selection acts on the codon usage bias. Our analysis of a large scale reveals new feature on DENV genomic evolution.

The role of cell proteins in dengue virus infection

UNLABELLED Despite 70 years of study, dengue disease continues to be a global health burden. Treatment is only supportive based on presenting symptoms. To date, there is no licensed prophylactic vaccine and no specific antiviral drugs available. The pathogenesis mechanisms during dengue virus infections remain poorly understood, and the complete picture on risk factors for developing severe clinical illness is still unknown. Viruses as obligate intracellular parasites depend on the host cell machinery for replication. As a result of a co-evolution process for million years, viruses have developed sophisticated strategies to hijack and use cellular factors for entry, replication and propagation, alternate host transmission and to combat host cell defenses. This review focuses on recent reports about cellular proteins involved along the dengue virus replication cycle, in prime cellular targets during the infection of both humans and mosquito hosts and also on the proteomics and other approaches that are being used to reveal the entire orchestration and most significant processes altered during infection. Identification of the key host cell factors involve in these processes will provide a better understanding of how viruses replicate and cause disease, and how to develop more effective therapeutic interventions. BIOLOGICAL SIGNIFICANCE Dengue disease is as a global health problem. The treatment is only supportive based on presenting symptoms. To date, there is no licensed prophylactic vaccine and no specific antiviral drugs available. The study of the interactions between virus and host cell proteins will provide a better understanding of how viruses replicate and cause disease. Here, we focus on the current knowledge about the cellular proteins involved during DENV infection in different target cells in the two hosts, mosquito and human.

Expression of Antimicrobial Peptides in Human Monocytic Cells and Neutrophils in Response to Dengue Virus Type 2.

Background/Aims: The innate immune response is remarkably important for controlling infections. Information about the participation of antimicrobial peptides (AMPs) in response to dengue virus (DENV) is scarce. The aim of this study was to examine the AMP response to DENV-2 in human THP-1 cells and neutrophils. Methods: Protein and mRNA levels of two AMPs - hBD-1 and cathelicidin LL-37 - were assessed in DENV-infected macrophage-like THP-1 cells using qRT-PCR and indirect immunofluorescence. Also, mRNA levels of α-defensins (hDEFAs) and LL-37 were examined by qRT-PCR in human neutrophils taken from peripheral blood and treated with DENV-2. Results: mRNA expression of hBD-1 rose in THP-1 cells at 24-72 h, while protein expression increased later, from 48 to 72 h after infection. Cathelicidin LL-37 mRNA expression of DENV-infected THP-1 cells was observed at 6-48 h after infection, while protein levels increased importantly up to 72 h after infection. Regarding neutrophils, the mRNA expression of hDEFAs and LL-37 increased slightly at 2 and 5 h after the contact with DENV-2. Conclusion: THP-1 cells and human neutrophils strongly respond to DENV by producing AMPs: hBD-1 and LL-37 for the THP-1 cells and hDEFAs and LL-37 for neutrophils. However, the direct effect of these molecules on DENV particles remains unclear.

Dengue Virus Type 2: Protein Binding and Active Replication in Human Central Nervous System Cells

An increased number of dengue cases with neurological complications have been reported in recent years. The lack of reliable animal models for dengue has hindered studies on dengue virus (DENV) pathogenesis and cellular tropism in vivo. We further investigate the tropism of DENV for the human central nervous system (CNS), characterizing DENV interactions with cell surface proteins in human CNS cells by virus overlay protein binding assays (VOPBA) and coimmunoprecipitations. In VOPBA, three membrane proteins (60, 70, and 130 kDa) from the gray matter bound the entire virus particle, whereas only a 70 kDa protein bound in white matter. The coimmunoprecipitation assays revealed three proteins from gray matter consistently binding virus particles, one clearly distinguishable protein (~32 kDa) and two less apparent proteins (100 and 130 kDa). Monoclonal anti-NS3 targeted the virus protein in primary cell cultures of human CNS treated with DENV-2, which also stained positive for NeuH, a neuron-specific marker. Thus, our results indicate (1) that DENV-2 exhibited a direct tropism for human neurons and (2) that human neurons sustain an active DENV replication as was demonstrated by the presence of the NS3 viral antigen in primary cultures of these cells treated with DENV-2.

Lack of Association of the Polymorphisms IL-17A (-197G/A) and IL-17F (+7488A/G) with Multibacillary Leprosy in Mexican Patients.

Background. Leprosy is a chronic infectious disease caused by the intracellular acid-fast bacilli Mycobacterium leprae; it has been determined that genetic factors of the host play an important role in the disease susceptibility. Thus, in this case-control study, we evaluated the possible association between the IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs and susceptibility to leprosy disease in Mexican population. Methods. Seventy-five leprosy patients and sixty-nine control subjects were included. Both SNPs were genotyped with the polymerase chain reaction-restriction fragment length polymorphism technique. Results. We found nonsignificant differences in genotype and allele frequencies related to IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780) gene SNPs in MB as well as subclinical forms of leprosy disease versus healthy individuals. Conclusions. Since the sample size is not large enough, it is difficult to sustain an association of susceptibility to leprosy with genotypes or allele frequencies of IL-17A G-197A (rs227593) and IL-17F A7488G (His161Arg, rs763780), suggesting that IL-17 polymorphisms have no significant role in the genetic susceptibility to development of this disease in the Mexican Mestizo population.

Detection of Hand, Foot and Mouth Disease in the Yucatan Peninsula of Mexico

We report a case of hand, foot and mouth disease (HFMD) in a 5-year-old male from Merida City in the Yucatan Peninsula of Mexico. A clinical and physical examination revealed that the patient had symptoms typical of HFMD, including fever, fatigue, odynophagia, throat edema, hyperemia, lesions on the hands and feet, and blisters in the oral cavity. The patient fully recovered after a convalescence period of almost three weeks. Reverse transcription-polymerase chain reaction and nucleotide sequencing revealed that the etiological agent was enterovirus 71 (EV71). The sequence has greatest (90.4%) nucleotide identity to the corresponding regions of EV71 isolates from the Netherlands and Singapore. Although HFMD is presumably common in Mexico, surprisingly there are no data in the PubMed database to support this. This case report provides the first peer-reviewed evidence of HFMD in Mexico.

Large Scale Genome Analysis Shows that the Epitopes for Broadly Cross-Reactive Antibodies Are Predominant in the Pandemic 2009 Influenza Virus A H1N1 Strain

The past pandemic strain H1N1 (A (H1N1)pdm09) has now become a common component of current seasonal influenza viruses. It has changed the pre-existing immunity of the human population to succeeding infections. In the present study, a total of 14,210 distinct sequences downloaded from National Center for Biotechnology Information (NCBI) database were used for the analysis. The epitope compositions in A (H1N1)pdm09, classic seasonal strains, swine strains as well as highly virulent avian strain H5N1, identified with the aid of the Immune Epitope DataBase (IEDB), were compared at genomic level. The result showed that A (H1N1) pdm09 contains the 90% of B-cell epitopes for broadly cross-reactive antibodies (EBCA), which is in consonance with the recent reports on the experimental identification of new epitopes or antibodies for this virus and the binding tests with influenza virus protein HA of different subtypes. Our analysis supports that high proportional EBCA depends on the epitope pattern of A (H1N1)pdm09 virus. This study may be helpful for better understanding of A (H1N1)pdm09 and the production of new influenza vaccines.

Dengue Virus Induces the Release of sCD40L and Changes in Levels of Membranal CD42b and CD40L Molecules in Human Platelets

Platelets are considered as significant players in innate and adaptive immune responses. The adhesion molecules they express, including P-selectin, CD40L, and CD42b, facilitate interactions with many cellular effectors. Upon interacting with a pathogen, platelets rapidly express and enhance their adhesion molecules, and secrete cytokines and chemokines. A similar phenomenon occurs after exposure of platelets to thrombin, an agonist extensively used for in vitro activation of these cells. It was recently reported that the dengue virus not only interacts with platelets but possibly infects them, which triggers an increased expression of adhesion molecule P-selectin as well as secretion of IL-1β. In the present study, surface molecules of platelets like CD40L, CD42b, CD62P, and MHC class I were evaluated at 4 h of interaction with dengue virus serotype 2 (DENV-2), finding that DENV-2 induced a sharp rise in the membrane expression of all these molecules. At 2 and 4 h of DENV-2 stimulation of platelets, a significantly greater secretion of soluble CD40L (sCD40L) was found (versus basal levels) as well as cytokines such as GM-CSF, IL-6, IL-8, IL-10, and TNF-α. Compared to basal, DENV-2 elicited more than two-fold increase in these cytokines. Compared to the thrombin-induced response, the level generated by DENV-2 was much higher for GM-CSF, IL-6, and TNF-α. All these events induced by DENV end up in conspicuous morphological changes observed in platelets by confocal microscopy and transmission electron microscopy, very different from those elicited by thrombin in a more physiological scenery.