A Simple Reverse Transcription-Polymerase Chain Reaction for Dengue Type 2 Virus Identification

We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identification of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50ml assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique

Polymerase chain reaction amplification of genomic fragments of bovine herpesvirus-1

Especial conditions were developed for the amplification of five DNA segments from US region of BHV-1 by polymerase chain reaction. In order to eliminate most nonspecific products it was found that addition of three cosolvents DMSO, glycerol and NP 40 was a simple method for increasing the specificity of amplification.

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.

Characterization of high osmolarity-induced vacuole fragmentation in "Saccharomyces c."

La majorité des organelles d'une cellule adaptent leur nombre et leur taille pendant les processus de division cellulaire, de trafic vésiculaire ou suite à des changements environnementaux par des processus de fusion et de fragmentation membranaires. Ceci est valable notamment pour le golgi, les mitochondries, les péroxisomes et les lysosomes. La vacuole est le compartiment terminal de la voie endocytaire dans la levure Saccharomyces cerevisiae\ elle correspond aux lysosomes des cellules mammifères. Suite à un choc hyperosmotique, la vacuole se fragmente en plusieurs petites vésicules. Durant ce projet, cette fragmentation a été étudiée en utilisant la technique de microscopie confocale in vivo. J'ai observé que la division de la vacuole se produit d'une façon asymétrique. La première minute après le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase est dépendante de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que d'un gradient transmembranaire de protons. Pendant les 10-15 minutes qui suivent, des vésicules se détachent dans les régions où l'on observe les invaginations pendant la phase initiale. Cette deuxième phase qui mène à la fission des nouveaux compartiments vacuolaires dépend de la production du lipide PI(3,5)P2 par la protéine Fab1. J'ai établi la suite des événements du processus de fragmentation des vacuoles et propose la possibilité d'un rôle régulateur de la protéine kinase cycline-dépendante Pho85.¦En outre, j'ai tenté d'éclaircir plus spécifiquement le rôle de Vps1 pendant la fusion et fission des vacuoles. J'ai trouvé que tous les deux processus sont dépendants de l'activité GTPase de cette protéine. De plus l'association avec la membrane vacuolaire paraît régulée par le cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'un autre facteur protéinique, ce qui permet de conclure à une interaction directe avec des lipides de la membrane. Cette interaction est au moins partiellement effectuée par le domaine GTPase, ce qui est une nouveauté pour un membre de cette famille de protéines. Une deuxième partie de Vps1, nommée insert B, est impliquée dans la liaison à la vacuole, soit par interaction directe avec la membrane, soit par régulation du domaine GTPase. En assumant que Vps1 détienne deux régions capables de liaison aux membranes, je conclus qu'elle pourrait fonctionner comme facteur de « tethering » lors de la fusion des vacuoles.¦-¦La cellule contient plusieurs sous-unités, appelées organelles, possédant chacune une fonction spécifique. Dépendant des processus qui s'y déroulent à l'intérieur, un environnement chimique spécifique est requis. Pour maintenir ces différentes conditions, les organelles sont séparées par des membranes. Lors de la division cellulaire ou en adaptation à des changements de milieu, les organelles doivent être capables de modifier leur morphologie. Cette adaptation a souvent lieu par fusion ou division des organelles. Le même principe est valable pour la vacuole dans la levure. La vacuole est une organelle qui sert principalement au stockage des aliments et à la dégradation des différents composants cellulaires. Alors que la fusion des vacuoles est un processus déjà bien décrit, la fragmentation des vacuoles a jusqu'ici été peu étudiée. Elle peut être induit par un choc osmotique: à cause de la concentration de sel élevé dans le milieu, le cytosol de la levure perd de l'eau. Par un flux d'eau de la vacuole au cytosol, la cellule est capable d'équilibrer celui-ci. Quand la vacuole perd du volume, elle doit réadapter le rapport entre surface membranaire et volume, ce qui se fait efficacement par une fragmentation d'une grande vacuole en plusieurs petites vésicules. Comment ce processus se déroule d'un point de vue morphologique n'a pas été décrit jusqu'à présent. En analysant la fragmentation vacuolaire par microscopie, j'ai trouvé que celle-ci se déroule en deux phases. Pendant la première minute suivant le choc osmotique, les vacuoles rétrécissent et forment des longues invaginations tubulaires. Cette phase dépend de la protéine Vps1, un membre de la famille des protéines apparentées à la dynamine, ainsi que du gradient transmembranaire de protons. Ce gradient s'établit par une pompe membranaire, la V-ATPase, qui transporte des protons dans la vacuole en utilisant l'énergie libérée par hydrolyse d'ATP. Après cette phase initiale, la formation de nouvelles vésicules vacuolaires dépend de la synthèse du lipide PI(3,5)P2.¦Dans la deuxième partie de l'étude, j'ai tenté de décrire comment Vps1 lie la membrane pour effectuer un remodelage de la vacuole. Vps1 est nécessaire pour la fusion et la fragmentation des vacuoles. J'ai découvert que tous les deux processus dépendent de sa capacité d'hydrolyser du GTP. Ainsi l'association avec la membrane est couplée au cycle d'hydrolyse du GTP. Vps1 peut lier la membrane sans la présence d'une autre protéine, et interagit donc très probablement avec les lipides de la membrane. Deux parties différentes de la protéine sont impliquées dans la liaison, dont une, inattendue, le domaine GTPase.¦-¦Numerous organelles undergo membrane fission and fusion events during cell division, vesicular traffic, or in response to changes in environmental conditions. Examples include Golgi (Acharya et al., 1998) mitochondria (Bleazard et al., 1999) peroxisomes (Kuravi et al., 2006) and lysosomes (Ward et al., 1997). In the yeast Saccharomyces cerevisiae the vacuole is the terminal component of the endocytic pathway and corresponds to lysosomes in mammalian cells. Yeast vacuoles fragment into multiple small vesicles in response to a hypertonic shock. This rapid and homogeneous reaction can serve as a model to study the requirements of the fragmentation process. Here, I investigated osmotically induced fragmentation by time-lapse microscopy. I observe that the small fragmentation products originate directly from the large central vacuole by asymmetric scission rather than by consecutive equal divisions and that fragmentation occurs in two distinct phases. During the first minute, vacuoles shrink and generate deep invaginations, leaving behind tubular structures. This phase requires the dynamin-like GTPase Vps1 and the vacuolar proton gradient. In the subsequent 10-15 minutes, vesicles pinch off from the tubular structures in a polarized fashion, directly generating fragmentation products of the final size. This phase depends on the production of phosphatidylinositol- 3,5-bisphosphate by the Fab1 complex. I suggest a possible regulation of vacuole fragmentation by the CDK Pho85. Based on my microscopy study I established a sequential involvement of the different fission factors.¦In addition to the morphological description of vacuole fragmentation I more specifically aimed to shed some light on the role of Vps1 in vacuole fragmentation and fusion. I find that both functions are dependent on the GTPase activity of the protein and that also the membrane association of the dynamin-like protein is coupled to the GTPase cycle. I found that Vps1 has the capacity for direct lipid binding on the vacuole and that this lipid binding is at least partially mediated through residues in the GTPase domain, a complete novelty for a dynamin family member. A second stretch located in the region of insert Β has also membrane-binding activity or regulates the association with the vacuole through the GTPase domain. Under the assumption of two membrane-binding regions I speculate on Vps1 as a possible tethering factor for vacuole fusion.

Relevé des consultations et des transferts de patients par enquête directe auprès des médecins: méthode d'une enquête, acceptabilité et limites d'interprétation. [Summary of consultations and transfer of patients by a direct survey of physicians: survey methods, acceptability and limits of interpretation].

A survey of medical ambulatory practice was carried out in February-March 1981 in the two Swiss cantons of Vaud and Fribourg (total population: 700,000), in which 205 physicians participated. The methodology used was inspired from the U.S. National Ambulatory Medical Care Survey, the data collection instrument of which was adapted to our conditions; in addition, data were gathered on all referrals prescribed by 154 physicians during two weeks. (The instruments used are presented.) The potential and limits of this type of survey are discussed, as well as the representativity of the participating physicians and of the recorded visits, which are a systematic sample of over 43,000 visits.

Sensitivity and specificity of polymerase chain reaction in Giemsa-stained slides for diagnosis of visceral leishmaniasis in children

The aim of this study was to evaluate the sensitivity and specificity of polymerase chain reaction (PCR) in the detection of Leishmania DNA in archived Giemsa-stained bone marrow slides for diagnosis of visceral leishmaniasis (VL), and to compare PCR with conventional diagnostic techniques, like direct microscopy and parasite culture. Specimens of archived Giemsa-stained bone marrow slides from 91 patients with VL and from 79 controls with other diseases or conditions were studied. PCR showed the highest sensitivity (92.3%) and had good specificity (97.5%). Direct examination detected 79.1% and culture 59% of positive samples. In addition, PCR was able to detect VL in 16 of 19 patients (84.2%) with negative microscopy. PCR in Giemsa-stained bone marrow slides is a suitable tool for confirming diagnosis in patients with VL and may be useful in the diagnosis of difficult cases. Slide smears are easily stored, do not require special storage conditions such as low temperatures, and can be easily mailed to centers where PCR is available, making it an excellent option for diagnosis in the field.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Gene transfer of interleukin-18-binding protein attenuates cardiac allograft rejection.

Interleukin (IL) 18 is a potent pro-inflammatory Th1 cytokine that exerts pleiotropic effector functions in both innate and acquired immune responses. Increased IL-18 production during acute rejection has been reported in experimental heart transplantation models and in kidney transplant recipients. IL-18-binding protein (IL-18BP) binds IL-18 with high affinity and neutralizes its biologic activity. We have analyzed the efficacy of an adenoviral vector expressing an IL-18BP-Ig fusion protein in a rat model of heart transplantation. IL-18BP-Ig gene transfer into Fisher (F344) rat donor hearts resulted in prolonged graft survival in Lewis recipients (15.8 +/- 1.4 days vs. 10.3 +/- 2.5 and 10.1 +/- 2.1 days with control virus and buffer solution alone, respectively; P < 0.001). Immunohistochemical analysis revealed decreased intra-graft infiltrates of monocytes/macrophages, CD4(+), CD8alpha(+) and T-cell receptor alphabeta(+) cells after IL-18BP-Ig versus mock gene transfer (P < 0.05). Real-time reverse transcriptase polymerase chain reaction analysis showed decreased cytokine transcripts for the RANTES chemokine and transforming growth factor-beta after IL-18BP-Ig gene transfer (P < 0.05). IL-18BP-Ig gene transfer attenuates inflammatory cell infiltrates and prolongs cardiac allograft survival in rats. These results suggest a contributory role for IL-18 in acute rejection. Further studies aiming at defining the therapeutic potential of IL-18BP are warranted.

Identification of SL addition trans-splicing acceptor sites in the internal transcribed spacer I region of pre-rRNA in Leishmania (Leishmania) amazonensis

Trypanosomatidae is a family of early branching eukaryotes harbouring a distinctive repertoire of gene expression strategies. Functional mature messenger RNA is generated via the trans-splicing and polyadenylation processing of constitutively transcribed polycistronic units. Recently, trans-splicing of pre-small subunit ribosomal RNA in the 5' external transcribed spacer region and of precursor tRNAsec have been described. Here, we used a previously validated semi-nested reverse transcription-polymerase chain reaction strategy to investigate internal transcribed spacer (ITS) I acceptor sites in total RNA from Leishmania (Leishmania) amazonensis. Two distinct spliced leader-containing RNAs were detected indicating that trans-splicing reactions occur at two AG acceptor sites mapped in this ITS region. These data provide further evidence of the wide spectrum of RNA molecules that act as trans-splicing acceptors in trypanosomatids.

Multiplex polymerase chain reaction to identify and determine the toxigenicity of Corynebacterium spp with zoonotic potential and an overview of human and animal infections

Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis constitute a group of potentially toxigenic microorganisms that are related to different infectious processes in animal and human hosts. Currently, there is a lack of information on the prevalence of disease caused by these pathogens, which is partially due to a reduction in the frequency of routine laboratory testing. In this study, a multiplex polymerase chain reaction (mPCR) assay that can simultaneously identify and determine the toxigenicity of these corynebacterial species with zoonotic potential was developed. This assay uses five primer pairs targeting the following genes: rpoB (Corynebacterium spp), 16S rRNA (C. ulcerans and C. pseudotuberculosis), pld (C. pseudotuberculosis), dtxR (C. diphtheriae) and tox [diphtheria toxin (DT) ]. In addition to describing this assay, we review the literature regarding the diseases caused by these pathogens. Of the 213 coryneform strains tested, the mPCR results for all toxigenic and non-toxigenic strains of C . diphtheriae, C. ulcerans and C. pseudotuberculosis were in 100% agreement with the results of standard biochemical tests and PCR-DT. As an alternative to conventional methods, due to its advantages of specificity and speed, the mPCR assay used in this study may successfully be applied for the diagnosis of human and/or animal diseases caused by potentially toxigenic corynebacterial species.

Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer.

Postischemic recovery of contractile function is better in hearts from fasted rats than in hearts from fed rats. In this study, we examined whether feeding-induced inhibition of palmitate oxidation at the level of carnitine palmitoyl transferase I is involved in the mechanism underlying impaired recovery of contractile function. Hearts isolated from fasted or fed rats were submitted to no-flow ischemia followed by reperfusion with buffer containing 8 mM glucose and either 0.4 mM palmitate or 0.8 mM octanoate. During reperfusion, oxidation of palmitate was higher after fasting than after feeding, whereas oxidation of octanoate was not influenced by the nutritional state. In the presence of palmitate, recovery of left ventricular developed pressure was better in hearts from fasted rats. Substitution of octanoate for palmitate during reperfusion enhanced recovery of left ventricular developed pressure in hearts from fed rats. However, the chain length of the fatty acid did not influence diastolic contracture. The results suggest that nutritional variation of mitochondrial fatty acid transfer may influence postischemic recovery of contractile function.

Induction of tolerogenic vs immunogenic dendritic cells (DCs) in the presence of GM-CSF is regulated by the strength of signaling from monophosphoryl lipid A (MPLA) in association with glutathione and fetal hemoglobin gamma-chain.

Previous studies showed a fetal sheep liver extract (FSLE), in association with monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS), could interact to induce the development of dendritic cells (DCs) which regulated production of Foxp3+ Treg. This interaction was associated with an altered gene expression both of distinct subsets of TLRs and of CD200Rs. Prior studies had suggested that major interacting components within FSLE were gamma-chain of fetal hemoglobin (Hgbgamma) and glutathione (GSH). We investigated whether differentiation/maturation of DCs in vitro in the presence of either GM-CSF or Flt3L to produce preferentially either immunogenic or tolerogenic DCs was itself controlled by an interaction between MPLA, GSH and Hgbgamma. At low (approximately 10 microg/ml) Hgbgamma concentrations, DCs developing in culture with GSH and MPLA produced optimal stimulation of allogeneic CTL cell responses in vitro (and enhanced skin graft rejection in vivo). At higher concentrations (>40 microg/ml Hgbgamma) and equivalent concentrations of MPLA and GSH, the DCs induce populations of Treg which can suppress the induction of allogeneic CTL and graft rejection in vivo. These different populations of DCs express different patterns of mRNAs for the CD200R family. Addition of anti-TLR or anti-MD-1 mAbs to DCs developing in this mixture (Hgbgamma+GSH+MPLA), suggests that one effect of (GSH+Hgbgamma) on MPLA stimulation may involve altered signaling through TLR4.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells.

To modulate alloreactivity after hematopoietic stem cell transplantation, "suicide" gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell-depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirus-mediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)-specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the "recent activation" phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA-CD27+CCR7- effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27-CCR7- terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27- cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.

Charge transfer in DNA: hole charge is confined to a single base pair due to solvation effects

We include solvation effects in tight-binding Hamiltonians for hole states in DNA. The corresponding linear-response parameters are derived from accurate estimates of solvation energy calculated for several hole charge distributions in DNA stacks. Two models are considered: (A) the correction to a diagonal Hamiltonian matrix element depends only on the charge localized on the corresponding site and (B) in addition to this term, the reaction field due to adjacent base pairs is accounted for. We show that both schemes give very similar results. The effects of the polar medium on the hole distribution in DNA are studied. We conclude that the effects of polar surroundings essentially suppress charge delocalization in DNA, and hole states in (GC)n sequences are localized on individual guanines