Combinatorial Anti-arenaviral Therapy with the Small Molecule SKI-1/S1P Inhibitor PF-429242 and Ribavirin

Arenaviruses are enveloped negative strand viruses that cause acute and chronic infections. Several Arenaviruses can cause severe hemorrhagic fever in humans. In West Africa Lassa virus causes several hundred thousand infections per year, while Junin, Machupo, Guanarito, and Sabia virus have emerged in South America. So far, only one drug is licensed against arenaviruses, the nucleoside analogue Ribavirin (Rib), which is effective when given early in disease, but shows only minor therapeutic effects in late stages of the infection. Previous works demonstrated that processing of the arenavirus glycoprotein precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexinisozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infectionand production of infectious virus. Recently, the SKI-1/S1P inhibitor PF-429242wasshownto inhibit Old World arenavirusGPCprocessing, cell-to-cell propagation, and infectious virus production. In the present study, we assessed the activity of PF-429242 against processing of the GPCs of the genetically and structurally more distant New World arenaviruses and found potent inhibition of processing of the GPCs of Junin, Machupo, and Guanarito virus. Using the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV), we studied the potency of PF-429242 in the context of acute and chronic infection. In line with published data, PF-429242 potently inhibited acute LCMV infection. PF-429242 was also highly active against chronic infection and drug treatment resulted in rapid extinction of the virus without emergence of drug-resistant variants. In a combinatorial drug approach, we found that PF-429242 potentiated the anti-viral effect of Rib in treatment of acute andchronic infection. Taken together, we showed that the SKI-1/S1P inhibitor PF-429242 is broadly active against GPC processing of all major human pathogenic arenaviruses. Apart from being potent in acute infection, the drug is remarkably active in clearing chronic infection and potentiated the anti-arenaviral activity of Rib.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Glucose transporter-1 deficiency syndrome: the expanding clinical and genetic spectrum of a treatable disorder.

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic diet.

Is (1→3)-β-D-glucan the missing link from bedside assessment to pre-emptive therapy of invasive candidiasis?

ABSTRACT: Invasive candidiasis is a frequent life-threatening complication in critically ill patients. Early diagnosis followed by prompt treatment aimed at improving outcome by minimizing unnecessary antifungal use remains a major challenge in the ICU setting. Timely patient selection thus plays a key role for clinically efficient and cost-effective management. Approaches combining clinical risk factors and Candida colonization data have improved our ability to identify such patients early. While the negative predictive value of scores and predicting rules is up to 95 to 99%, the positive predictive value is much lower, ranging between 10 and 60%. Accordingly, if a positive score or rule is used to guide the start of antifungal therapy, many patients may be treated unnecessarily. Candida biomarkers display higher positive predictive values; however, they lack sensitivity and are thus not able to identify all cases of invasive candidiasis. The (1→3)-β-D-glucan (BG) assay, a panfungal antigen test, is recommended as a complementary tool for the diagnosis of invasive mycoses in high-risk hemato-oncological patients. Its role in the more heterogeneous ICU population remains to be defined. More efficient clinical selection strategies combined with performant laboratory tools are needed in order to treat the right patients at the right time by keeping costs of screening and therapy as low as possible. The new approach proposed by Posteraro and colleagues in the previous issue of Critical Care meets these requirements. A single positive BG value in medical patients admitted to the ICU with sepsis and expected to stay for more than 5 days preceded the documentation of candidemia by 1 to 3 days with an unprecedented diagnostic accuracy. Applying this one-point fungal screening on a selected subset of ICU patients with an estimated 15 to 20% risk of developing candidemia is an appealing and potentially cost-effective approach. If confirmed by multicenter investigations, and extended to surgical patients at high risk of invasive candidiasis after abdominal surgery, this Bayesian-based risk stratification approach aimed at maximizing clinical efficiency by minimizing health care resource utilization may substantially simplify the management of critically ill patients at risk of invasive candidiasis.

Increased expression of monocarboxylate transporters 1, 2, and 4 in colorectal carcinomas.

Tumour cells are known to be highly glycolytic, thus producing high amounts of lactic acid. Monocarboxylate transporters (MCTs), by promoting the efflux of the accumulating acids, constitute one of the most important mechanisms in the maintenance of tumour intracellular pH. Since data concerning MCT expression in colorectal carcinomas (CRC) are scarce and controversial, the present study aimed to assess the expressions of MCT1, 2, and 4 in a well characterized series of CRC and assess their role in CRC carcinogenesis. CRC samples (126 cases) were analyzed for MCT1, MCT2, and MCT4 immunoexpression and findings correlated with clinico-pathological parameters. Expression of all MCT isoforms in tumour cells was significantly increased when compared to adjacent normal epithelium. Remarkably, there was a significant gain of membrane expression for MCT1 and MCT4 and loss of plasma membrane expression for MCT2 in tumour cells. Plasma membrane expression of MCT1 was directly related to the presence of vascular invasion. This is the larger study on MCT expression in CRC and evaluates for the first time its clinico-pathological significance. The increased expression of these transporters suggests an important role in CRC, which might justify their use, especially MCT1 and MCT4, as targets in CRC drug therapy.

Altruism across disciplines: one word, multiple meanings

Altruism is a deep and complex phenomenon that is analysed by scholars of various disciplines, including psychology, philosophy, biology, evolutionary anthropology and experimental economics. Much confusion arises in current literature because the term altruism covers variable concepts and processes across disciplines. Here we investigate the sense given to altruism when used in different fields and argumentative contexts. We argue that four distinct but related concepts need to be distinguished: (a) psychological altruism, the genuine motivation to improve others' interests and welfare; (b) reproductive altruism, which involves increasing others' chances of survival and reproduction at the actor's expense; (c) behavioural altruism, which involves bearing some cost in the interest of others; and (d) preference altruism, which is a preference for others' interests. We show how this conceptual clarification permits the identification of overstated claims that stem from an imprecise use of terminology. Distinguishing these four types of altruism will help to solve rhetorical conflicts that currently undermine the interdisciplinary debate about human altruism.

Reduction in the expression of glucose transporter protein GLUT 2 in preneoplastic and neoplastic hepatic lesions and reexpression of GLUT 1 in late stages of hepatocarcinogenesis.

Expression of two important glucose transporter proteins, GLUT 2 (which is the typical glucose transporter in hepatocytes of adult liver) and the erythroid/brain type glucose transporter GLUT 1 (representing the typical glucose transporter in fetal liver parenchyma), was studied immunocytochemically during hepatocarcinogenesis in rats at different time points between 7 and 65 wk after cessation of 7-wk administration of 12 mg/kg of body weight of N-nitrosomorpholine p.o. (stop model). Foci of altered hepatocytes excessively storing glycogen (GSF) and mixed cell foci (MCF) composed of both glycogenotic and glycogen-poor cells were present at all time points studied. Seven wk after withdrawal of the carcinogen, GSF were the predominant type of focus of altered hepatocytes. Morphometrical evaluation of the focal lesions revealed that the number and volume fraction of GSF increased steadily until Wk 65. MCF were rare at 7 wk, increased slightly in number and size until Wk 37, but showed a pronounced elevation in their number and volume fraction from Wk 37 to Wk 65. In both GSF and MCF, GLUT 2 was generally decreased or partially absent at all time points. Consequently, foci of decreased GLUT 2 expression showed a steady increase in number and volume fraction from Wk 7 to Wk 65. GLUT 1 was lacking in GSF but occurred in some MCF from Wk 50 onward. The liver type glucose transporter GLUT 2 was decreased in all adenomas and hepatocellular carcinomas (HCC). In three of seven adenomas and 10 of 12 carcinomas, expression of GLUT 1 was increased compared with normal liver parenchyma. In two cases of adenoid HCC, cells of ductular formations coexpressed GLUT 2 and GLUT 1. In contrast, normal bile ducts, bile duct proliferations, and cystic cholangiomas expressed only GLUT 1. Seven of 12 HCC contained many microvessels intensely stained for GLUT 1, a phenomenon never observed in normal liver. Whenever adenoid tumor formations occurred, GLUT 1-positive microvessels were located in the immediate vicinity of these formations. Only in one HCC were such microvessels found in the absence of adenoid formations. Our studies indicate that a reduction of GLUT 2 expression occurs already in early preneoplastic hepatic foci and is maintained throughout hepatocarcinogenesis, including benign and malignant neoplasms. Reexpression of GLUT 1, however, appears in a few MCF and in the majority of adenomas and carcinomas.

Human immunodeficiency virus type 1: drug resistance in treated and untreated Brazilian children

Twenty-two vertically human immunodeficiency virus type 1 (HIV-1) infected Brazilian children were studied for antiretroviral drug resistance. They were separated into 2 groups according to the administration of antiretroviral therapy into those who presented disease symptoms or without symptoms and no therapy. Viral genome sequencing reactions were loaded on an automated DNA sampler (TruGene, Visible Genetics) and compared to a database of wild type HIV-1. In the former group 8 of 12 children presented isolates with mutations conferring resistance to protease inhibitors (PIs), 7 presented isolates resistant to nucleoside reverse transcriptase inhibitors (NRTIs) and 2 presented isolates resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten children were included in the antiretroviral naïve group. Eight were susceptible to NRTIs and all of them were susceptible to PIs; one presented the V108I mutation, which confers low-level resistance to NNRTIs. The data report HIV mutant isolates both in treated and untreated infants. However, the frequency and the level of drug resistance were more frequent in the group receiving antiretroviral therapy, corroborating the concept of selective pressure acting on the emergence of resistant viral strains. The children who presented alterations at polymorphism sites should be monitored for the development of additional mutations occurring at relevant resistance codons.

Biocontrol by phenazine-1-carboxamide-producing Pseudomonas chlororaphis PCL1391of tomato root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici

Seventy bacterial isolates from the rhizosphere of tomato were screened for antagonistic activity against the tomato foot and root rot-causing fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici. One isolate, strain PCL1391, appeared to be an efficient colonizer of tomato roots and an excellent biocontrol strain in an F. oxysporum/tomato test system. Strain PCL1391 was identified as Pseudomonas chlororaphis and further characterization showed that it produces a broad spectrum of antifungal factors (AFFs), including a hydrophobic compound, hydrogen cyanide, chitinase(s), and protease(s). Through mass spectrometry and nuclear magnetic resonance, the hydrophobic compound was identified as phenazine-1-carboxamide (PCN). We have studied the production and action of this AFF both in vitro and in vivo. Using a PCL1391 transposon mutant, with a lux reporter gene inserted in the phenazine biosynthetic operon (phz), we showed that this phenazine biosynthetic mutant was substantially decreased in both in vitro antifungal activity and biocontrol activity. Moreover, with the same mutant it was shown that the phz biosynthetic operon is expressed in the tomato rhizosphere. Comparison of the biocontrol activity of the PCN-producing strain PCL1391 with those of phenazine-1-carboxylic acid (PCA)-producing strains P. fluorescens 2-79 and P. aureofaciens 30-84 showed that the PCN-producing strain is able to suppress disease in the tomato/F. oxysporum system, whereas the PCA-producing strains are not. Comparison of in vitro antifungal activity of PCN and PCA showed that the antifungal activity of PCN was at least 10 times higher at neutral pH, suggesting that this may contribute to the superior biocontrol performance of strain PCL1391 in the tomato/F. oxysporum system.

Alcohol MOT part one

The Alcohol MOT is designed to support those working in primary care to carry out alcohol brief interventions. There is extensive evidence to show that primary care-based brief interventions are very effective at reducing drinking at both hazardous and harmful levels. MOT Part 1 enables patients to work out if they are drinking at hazardous or harmful levels. MOT Part 2 helps motivate and support patients to reduce their drinking. Both tools are designed so that a practitioner can work through them with a patient, or a patient can work through them alone.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Human immunodeficiency virus type 1 Brazilian subtype B variant showed an increasing avidity of the anti-V3 antibodies over time compared to the subtype B US/European strain in São Paulo, Brazil

The Brazilian variant of human immunodeficiency virus type 1 (HIV-1) subtype B, (serotype B"-GWGR), has a tryptophan replacing the proline in position 328 the HIV-1 envelope. A longer median time period from infection to acquired immunodeficiency syndrome (AIDS) for serotype B (B"-GWGR) infected subjects compared to the B-GPGR US/European strain was reported. In a cohort study, in São Paulo city, 10 B"-GWGR patients had a statistically significant increased avidity of the anti-V3 antibodies, from 79% ± 33% to 85% ± 75%, versus from 48% ± 59% to 32% ± 17% for the 10 B-GPGR subjects (p = 0.02). The T CD4+ cells showed a mean increase of + 0.45 cells/month for the B-GPGR subjects and for B"-GWGR the slope was + 1.24 cells/month (p = 0.06), for 62 and 55 months of follow up, respectively. RNA plasma viral load decreased from 3.98 ± 1.75 to 2.16 ± 1.54 log10 in the B"-GWGR group while B-GPGR patients showed one log10 reduction in viral load from 4.09 ± 0.38 to 3.17 ± 1.47 log10 over time (p = 0.23), with a decreasing slope of 0.0042 ± log10,/month and 0.0080 ± log10/month, for B-GPGR and B"-GWGR patients, respectively (p = 0.53). Neither group presented any AIDS defining events during the study, according to Center for Diseases Control criteria. Although the sample size is small, these results may indicate that differences in the pathogenicity of the 2 HIV-1 B serotypes which co-circulate in Brazil may be correlated to the avidity of anti-V3 antibodies.

Detection of cytotoxic necrotizing factor types 1 and 2 among fecal Escherichia coli isolates from brazilian children with and without diarrhea

The enteropathogenic role of cytotoxic necrotizing factor (CNF)-producing Escherichia coli was investigated by searching cnf genes among 2074 isolates from 200 children with and 200 without acute diarrhea in Brazil. Fourteen (7%) cases versus 10 (5%) control children carried at least one cnf positive isolate (P = 0.50) and most isolates expressed CNF type 1. DNA sequences of virulence factors of extraintestinal pathogenic E. coli (ExPEC) were detected in 78.6% of CNF1-producing isolates. Besides not being associated with human acute diarrhea, the CNF1-producing isolates here identified may represent potential ExPEC transitorily composing the normal intestinal flora.

Stimulation-induced increases of astrocytic oxidative metabolism in rats and humans investigated with 1-11C-acetate

The purpose of this study was to investigate astrocytic oxidative metabolism using 1-(11)C-acetate. 1-(11)C-acetate kinetics were evaluated in the rat somatosensory cortex using a beta-scintillator during different manipulations (test-retest, infraorbital nerve stimulation, and administration of acetazolamide or dichloroacetate). In humans a visual activation paradigm was used and kinetics were measured with positron emission tomography. Data were analyzed using a one-tissue compartment model. The following features supported the hypothesis that washout of radiolabel (k(2)) is because of (11)C-CO(2) and therefore related to oxygen consumption (CMRO(2)): (1) the onset of (11)C washout was delayed; (2)k(2) was not affected by acetazolamide-induced blood flow increase; (3)k(2) demonstrated a significant increase during stimulation in rats (from 0.014+/-0.007 to 0.027+/-0.006 per minute) and humans (from 0.016+/-0.010 to 0.026+/-0.006 per minute); and (4) dichloroacetate led to a substantial decrease of k(2). In the test-retest experiments K(1) and k(2) were very stable. In summary, 1-(11)C-acetate seems a promising tracer to investigate astrocytic oxidative metabolism in vivo. If the washout rate indeed represents the production of (11)C-CO(2), then its increase during stimulation would point to a substantially higher astrocytic oxidative metabolism during brain activation. However, the quantitative relationship between k(2) and CMRO(2) needs to be determined in future experiments.

Antiretroviral resistance and genetic diversity of human immunodeficiency virus type 1 isolates from the Federal District, Central Brazil

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasília, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).