Reliable resolution of ambiguous hepatitis C virus genotype 1 results with the Abbott HCV Genotype Plus RUO assay.

Accurate subtyping of hepatitis C virus genotype 1 (HCV-1) remains clinically and epidemiologically relevant. The Abbott HCV Genotype Plus RUO (GT Plus) assay, targeting the core region, was evaluated as a reflex test to resolve ambiguous HCV-1 results in a challenging sample collection. 198 HCV-1 specimens were analysed with GT Plus (38 specimens with and 160 without subtype assigned by the Abbott RealTime Genotype II (GT II) assay targeting the 5'NC and NS5B regions). Sanger sequencing of the core and/or NS5B regions were performed in 127 specimens without subtype assignment by GT II, with "not detected" results by GT Plus, or with mixed genotypes/subtypes. The remaining GT Plus results were compared to LiPA 2.0 (n = 45) or just to GT II results if concordant (n = 26). GT Plus successfully assigned the subtype in 142/160 (88.8%) samples. "Not detected" results indicated other HCV-1 subtypes/genotypes or mismatches in the core region in subtype 1b. The subtyping concordance between GT Plus and either sequencing or LiPA was 98.6% (140/142). Therefore, combined use of GT II and GT Plus assays represents a reliable and simple approach which considerably reduced the number of ambiguous HCV-1 results and enabled a successful subtyping of 98.9% of all HCV-1 samples.

Comparison of eotaxin-3 biomarker in patients with eosinophilic oesophagitis, proton pump inhibitor-responsive oesophageal eosinophilia and gastro-oesophageal reflux disease.

BACKGROUND: Proton pump inhibitor-responsive oesophageal eosinophilia (PPI-REE) is a recently described entity which resembles oeosinophilic oesophagitis (EoE), yet responds to acid suppressive treatment. AIM: To determine whether EoE shares similar staining features with PPI-REE or with gastro-oesophageal reflux disease (GERD). METHODS: This retrospective study consisted of patients with an established diagnosis of EoE, PPI-REE, or GERD identified from a database during a 1-year period. Immunohistochemistry (IHC) analysis was performed specifically targeting eotaxin-3 antibodies. All sections were qualitatively (intensity) and quantitatively (percentage of cells stained) assessed independently by two blinded pathologists. RESULTS: The cohort consisted of three groups of patients: EoE (n = 22), PPI-REE (n = 23) and GERD (n = 23) for a total of 68 patients. Study demographics included mean age 39 (14) years, 75% male and 77% Caucasian. There was a significant difference in the eotaxin-3 staining among EoE, PPI-REE and GERD groups [mean score (s.d.): 1.2 (1.2), 0.8 (1.0), 0.3 (0.7), P = 0.006]. Staining scores of EoE patients were significantly higher compared with GERD (P = 0.002) and a trend towards significance was seen between EoE and PPI-REE (P = 0.054). There was also a significant difference in EoE staining intensity score among the three groups (P = 0.006). Intensity scores of EoE were significantly higher compared with GERD [1.0 (0.9) vs. 0.22 (0.52), P < 0.001]. There was no significant difference between EoE and PPI-REE groups [1.0 (0.0) vs. 0.52 (0.75) P = 0.094]. CONCLUSIONS: A difference in eotaxin-3 staining was seen in the three groups of patients with oesophageal eosinophilia. Eotaxin-3 can distinguish EoE from GERD, but not from proton pump inhibitor responsive-oesophageal eosinophilia.

Household transmissibility and other characteristics of seasonal oseltamivir-resistant influenza A(H1N1) viruses, Germany, 2007-8.

During the influenza season 2007-8, the proportion of seasonal influenza A(H1N1) viruses resistant to the neuraminidase inhibitor oseltamivir increased worldwide. We conducted an investigation to compare patients infected with oseltamivir-resistant (ose-R) and oseltamivir- susceptible (ose-S) influenza A(H1N1) viruses regarding risk factors for resistance and the capability to transmit in the household setting. Within a cohort of 396 laboratory confirmed influenza patients from sentinel physicians we conducted a nested case-control study among patients infected with A(H1N1). Thirty patients in the cohort were infected with influenza B, none with influenza A(H3N2) and 366 with A(H1N1). Of the 366 A(H1N1) viruses 52 (14%) were ose-R. Demographic characteristics, oseltamivir exposure, travel history and outcome were not significantly different between ose-S and ose-R patients. Among 133 households in the nested case-control study, secondary household attack rates in households with ose-R cases and households with ose-S cases were similar (23 versus 26%; p-value=0.54). Ose-R household status and occurrence of secondary cases were associated with an odds ratio of 0.85 (95% confidence interval 0.38-1.88). We conclude that seasonal ose-R influenza A(H1N1) viruses have transmitted well in the household setting.

Coração de atleta e cardiomiopatia hipertrófica: é possível diferenciar? / Athlete's heart and hypertrophic cardiomyopathy: is it possible to differentiate?

A doença arterial coronariana é uma das principais causas de morbidades no mundo. O tratamento clínico, associado à mudança de estilo de vida, é sua base terapêutica. Somando-se a isso, para evitar a progressão da doença arterial coronariana, melhorar a qualidade de vida e reduzir a intensidade da angina, deve-se considerar a revascularização miocárdica. As indicações da cirurgia de revascularização miocárdica tem passado por mudanças, desde que a primeira intervenção cirúrgica foi realizada em 1967. Nesse texto é feita a revisão da literatura com base nos estudos que avaliaram as indicações cirúrgicasde revascularização miocárdica na doença arterial crônica, enfocando, principalmente, aqueles que compararam a abordagem cirúrgica com o tratamento clínico...

Prodrugs of perzinfotel with improved oral bioavailability.

Previous studies with perzinfotel (1), a potent, selective, competitive NMDA receptor antagonist, showed it to be efficacious in inflammatory and neuropathic pain models. To increase the low oral bioavailability of 1 (3-5%), prodrug derivatives (3a-h) were synthesized and evaluated. The oxymethylene-spaced diphenyl analogue 3a demonstrated good stability at acidic and neutral pH, as well as in simulated gastric fluid. In rat plasma, 3a was rapidly converted to 1 via 2a. Pharmacokinetic studies indicated that the amount of systemic exposure of 1 produced by a 10 mg/kg oral dose of 3a was 2.5-fold greater than that produced by a 30 mg/kg oral dose of 1. Consistent with these results, 3a was significantly more potent and had a longer duration of activity than 1 following oral administration in a rodent model of inflammatory pain. Taken together, these results demonstrate that an oxymethylene-spaced prodrug approach increased the bioavailability of 1.

Hepatitis C virus E2 envelope protein induces dendritic cell maturation.

Maturation is a critical process for dendritic cells (DC) to gain or enhance their functions in antigen presentation and T-cell activation. In this study, we investigated the effect of hepatitis C virus (HCV) envelope protein E2 on DC maturation and related functions. We show that binding of E2 protein to DC leads to a change from immature to mature phenotype as detected by an increased expression of cell surface molecules including CD83, CD80, CD86, CD11c and MHC class II. The E2-matured DC showed higher capacity to stimulate T-cell proliferation and interferon-gamma production and displayed higher levels of interleukin-12 production when compared with immature DC. The induction of DC maturation by E2 is both time- and dose-dependent and can be inhibited by anti-E2 antibodies. In addition, DC matured by E2 showed decreased uptake of bovine serum albumin and latex beads, indicating their decreased activities of endocytosis and phagocytosis upon maturation. Taken together, our results demonstrated that E2 protein is able to induce dendritic cell maturation and suggested that E2 protein may play an important role in regulation of immune responses during HCV infection.

Tuberculose cutânea disseminada com escrofuloderma associado à tuberculose de arco costal / Disseminated cutaneous tuberculosis with scrofuloderma associated to costal arch tuberculosis

Os autores relatam caso de tuberculose cutânea disseminada com escrofuloderma associado à tuberculose de arco costal. Paciente de 46 anos, do sexo feminino, há um ano com nódulos de um a 6cm em região cervical, dorso, axilas e regiões glúteas, que culminavam com fistulização e eliminação de secreção purulenta, associados a febre vespertina diária, sudorese noturna e emagrecimento de 10kg nos últimos três meses. A radiografia de tórax mostrou lesão lítica na terceira costela esquerda. A cultura de secreção do nódulo foi positiva para Mycobacterium tuberculosis. O tratamento para tuberculose resultou em melhora clínica e resolução das lesões cutâneas da paciente.

The authors describe a case of disseminated cutaneous tuberculosis with scrofuloderma associated to tuberculosis in the costal arch. A 46-year-old, woman, was hospitalized with nodules measuring 1 to 6 cm in the cervical region, back, armpit and buttocks, which developed purulent discharge with elimination of caseous secretion, associated to episodes of daily evening fever, night sweats and weight loss of 10Kg over the previous three months. Thorax radiography showed an osteolytic lesion in the third left rib. Culture of the nodule secretion was positive for Mycobacterium tuberculosis. Treatment for tuberculosis resulted in pronounced clinical improvement and resolution of the patient's cutaneous lesions.

Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF starvation leads to apoptosis of some of the ES-derived differentiated cells, together with p38alpha mitogen-activated protein kinase (MAPK) activation. Apoptosis, but not morphological cell differentiation, is blocked by a p38 inhibitor, PD169316. To further understand the mechanism of action of this compound, we have identified its specific targets by microarray studies. We report on the global expression profiles of genes expressed at 3 days upon LIF withdrawal (d3) compared to pluripotent cells and of genes whose expression is modulated at d3 under anti-apoptotic conditions. We showed that at d3 without LIF cells express, earlier than anticipated, specialized cell markers and that when the apoptotic process was impaired, expression of differentiation markers was altered. In addition, functional tests revealed properties of anti-apoptotic proteins not to alter cell pluripotency and a novel role for metallothionein 1 gene, which prevents apoptosis of early differentiated cells.

A p38 inhibitor allows to dissociate differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells.

Mouse embryonic stem cells remain pluripotent when maintained in the presence of leukemia inhibitory factor (LIF). Upon LIF withdrawal, most cells differentiate into various lineages, while some die by apoptosis within 3 days. We have analyzed the activation pattern of the mitogen-activated protein kinase (MAPK) families and characterized the expression profile of selected genes modulated during differentiation or apoptosis. We show that p38 MAPKs are activated first, during the apoptotic crisis, while extracellular-regulated kinases and c-Jun N-terminal kinases are induced after the apoptotic crisis in differentiated cells. However, by using both p38 kinase inhibitors (PD169316 and SB203580) and a p38alpha(-/-) cell line, we demonstrate that p38alpha activation is rather a consequence than a cause of apoptosis. We thus reveal novel properties of PD169316, which induces cell survival without impairing cell differentiation, and identify PD169316-sensitive targets like the fibroblast growth factor-5, Brachyury and bcl-2 genes. Finally, we demonstrate that overexpression of the PD169316 - regulated bcl-2 gene prevents LIF withdrawal - induced cell death.

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

Intensity of vascular endothelial growth factor expression is associated with increased risk of recurrence and decreased disease-free survival in papillary thyroid cancer.

BACKGROUND: Vascular endothelial growth factor (VEGF) induces proliferation of endothelial cells, stimulates angiogenesis, and increases vascular permeability. Increased VEGF expression has been associated with poor clinical outcomes in many malignancies. Several recent reports have documented over expression of VEGF in papillary thyroid cancer. We hypothesized that increased expression of VEGF would be associated with either an increased risk of recurrence or a decreased recurrence-free survival in papillary thyroid cancer. METHODS: Immunohistochemistry was used to detect VEGF expression in archival paraffin-embedded surgical thyroid specimens from 96 subjects with papillary thyroid cancer. RESULTS: VEGF expression was detected in 98% (94/96) of the samples, predominantly of slight-to-moderate intensity in the majority of malignant cells. However, the specific finding of a diffuse pattern of intense immunostaining for VEGF was detected significantly more often than less intense, patchy immunostaining patterns in subjects with distant metastasis at diagnosis (63% versus 15%, P =.005), local recurrence (58% versus 13%, P =.001), and distant recurrence (83% versus 14%, P =.001). Furthermore, this specific pattern of diffuse, intense VEGF expression was associated with a significantly shorter recurrence-free survival than other staining patterns (P =.007). CONCLUSIONS: These data demonstrate that the immunohistochemical pattern of VEGF staining in the initial surgical specimen is strongly associated with the incidence of local and distant metastasis in papillary thyroid cancer.

High-density optical data storage with one-photon and two-photon near-field fluorescence microscopy.

A spatially localized photochemical reaction induced by near-field femtosecond laser pulses is demonstrated on a nanometer scale and used for high-density optical data storage. Recorded domains down to 120 and 70 nm are obtained with one-photon and two-photon excitation, respectively. It is shown that the local-field confinement that is due to the quadratic dependence of two-photon excitation on light intensity has the potential to increase the near-field optical storage density.

Role of suppressors of cytokine signaling (Socs) in leukemia inhibitory factor (LIF) -dependent embryonic stem cell survival.

Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF). LIF withdrawal results in progressive ES cell differentiation. Here we show that during this differentiation process, part of the cells undergo apoptosis concomitant with an activation of the p38 MAP kinase. To gain insight into events mediated by LIF in ES cells, the expression of potential candidate genes was analyzed in the absence or presence of this cytokine by using a semiquantitative RT-PCR assay. We focused on early response genes and on a new type of cytokine repressors (the Socs proteins), some of which exhibit anti-apoptotic properties. We found that expression of c-Fos, c-Jun, and JunB was induced upon LIF treatment whereas that of JunD, the tyrosine phosphatase ESP, and the components of the LIF receptor remained unaffected. Expression of Socs-3, but not Socs-1 or Socs-2, was stimulated in the presence of LIF. Finally, uncontrolled overexpression of Socs-1 and Socs-3 led to repression of LIF-dependent transcription and severely reduced cell viability, suggesting that the disturbance of a well balanced Socs protein content has adverse effects on cell survival.

Medication assistance programs for uninsured and indigent patients.

Pharmaceutical company-sponsored medication assistance programs and the pharmacist's role in obtaining medications for indigent patients are discussed. Information about enrolling in medication assistance programs can be obtained through a variety of sources, including the Internet. Although some eligibility criteria may be common to many programs, each company operates independently, and the eligibility criteria vary, individuals involved in the administration of medication assistance programs should strive to ensure that correct information is reported. Pharmacists can play an important role in acquiring medications for patients by reviewing patient information, recommending patients for enrollment, and serving as a liaison between the patient or health care provider and the program administrator. Many pharmaceutical companies have programs that provide prescription medications to indigent or uninsured patients. Pharmacists can serve as a liaison between these programs and the patient or the health care provider.

The relevance of plasminogen activator inhibitor 1 (PAI-1) as a marker for the diagnosis of hepatic veno-occlusive disease in patients after bone marrow transplantation.

Hepatic veno-occlusive disease (VOD) is the third most important fatal complication in allogeneic bone marrow transplantation (BMT), the second most significant one in the autologous setting and the most severe of all the regimen related toxicities. A growing number of VOD cases has to be expected due to the increasing number of high dose chemotherapies given with consecutive stem cell transplantation in patients with solid tumors. Confirmation of the diagnosis of VOD by biopsy is associated with a high risk of severe bleeding complications and, unfortunately, until now reliable laboratory markers have not as yet been established. Recently, plasminogen activator inhibitor 1 (PAI-1), the main inhibitor of the fibrinolytic system, has been found to be significantly elevated in VOD patients probably reflecting hypofibrinolysis in these patients. Furthermore, PAI-1 was able to distinguish between patients with VOD and those with hyperbilirubinemia after BMT caused by graft-versus-host-disease (GVHD) or toxic effects, in which cases the PAI-1 levels were mostly within the normal range. In this overview we summarize the data strongly indicating that PAI-1 is a useful marker for the diagnosis of VOD and helps in the differential diagnosis of hyperbilirubinemia after BMT.

Altered expression of extracellular matrix in human-cytomegalovirus-infected cells and a human artery organ culture model to study its biological relevance.

The influence of human cytomegalovirus (HCMV) on the transcription of 11 selected, representative extracellular matrix genes was investigated in cell culture. Northern blot hybridization indicated the downregulation of all mRNAs investigated. Based on our results and the known repression of other extracellular matrix transcripts and the beta-actin transcription during HCMV infection, we suggest that one molecular mechanism contributing to the cytopathic effect may be the transcriptional downregulation of genes encoding proteins involved in cell structure and intercellular connection. To further study the biological relevance of this and other pathogenetic mechanisms, we established a human renal artery organ culture system and characterized this new infection model for HCMV. Our model is a new suitable system for the investigation of molecular as well as functional consequences of HCMV infection in a more physiological microenvironment.

Development of viremia and humoral and cellular parameters of immune activation after vaccination with yellow fever virus strain 17D: a model of human flavivirus infection.

To monitor early and late events of immune system activation after primary and secondary flavivirus infection, 17 healthy persons were vaccinated with the standard 17D vaccine virus strain of yellow fever (YF). Twelve of these persons had not received YF vaccine previously and 5 had been vaccinated once at least 10 years before. Viremia and various parameters of humoral and cellular immune activation were followed daily for 7 days and weekly thereafter. Viremia was detected by reverse transcriptase-polymerase chain reaction in all 12 first-time vaccinees beginning from the second to the sixth day after vaccination; most tested positive between the fourth and sixth day. Infectious 17D virus was detected using a plaque forming assay in the serum of 7 of the 12 first-time vaccinees. As first parameters of immune activation, neopterin and beta2-microglobulin markedly increased between day 2 and day 6 postvaccination. In parallel to the viremia, circulating CD8+ T-cells significantly increased, with peak levels at day 5 after primary vaccination, indicating an activation of the cellular immune system. Neither viremia nor significant changes of these activation markers were observed in the five revaccinated persons. Neutralizing antibodies directed against the 17D vaccine strain developed in all persons within 2 weeks after vaccination. No correlation was found between the extent of viremia and the titer of neutralizing antibodies. Revaccination was followed by a minor and transient increase of neutralizing antibodies. High titers of neutralizing antibodies persisted for at least 10 years after primary vaccination.