Journal articles: 'Roches salines' – Grafiati (2024)

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As atividades de projeto e instalação de colunas de revestimento em poços de petróleo correspondem a elementos fundamentais para garantir a segurança e a operacionalização de campos exploratórios. Com o desafio da perfuração de poços em áreas geologicamente mais complexas, a exemplo das chamadas reservas do pré-sal e em reservatórios sujeitos à alta pressão e alta temperatura (High Pressure High Temperature - HPHT), novas variáveis de projeto devem ser consideradas. Este trabalho propõe-se a avaliar o comportamento de modelos constitutivos de rochas salinas por meios de curvas de fluências objetivando avaliar o impacto do efeito de fluência da rocha salina na estabilidade do poço aberto na fase de perfuração. Também são avaliados os incrementos de esforços advindos do comportamento viscoso das rochas salinas nas colunas de revestimento de poços de petróleo e a respectiva influência no tratamento de projetos para dimensionamento de revestimento de poços. Emprega-se uma estratégia baseada no método dos elementos finitos para modelar o comportamento de fluência em rochas salinas através de modelos viscoelásticos em função da série de Pronny. O processo de dimensionamento das colunas de revestimento é efetuado seguindo as recomendações normativas da API5C3 e ISO10400.

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The taxonomic structure of soil microbial community was studied in six samples taken from a salt marsh along the salinity gradient and in two samples of non-saline soils using pyrosequencing method (454 Roche). The analysis allowed to identify three main ecological groups of microorganisms depending on the degree of the soil salinity. Halophylic microorganisms were mainly represented by bacteria of three phyla Firmicutes, Proteobacteria and Bacteroidetes and included much less of archaea (the Halobacteriaceae family). Within the distance of 150–200 m from the point with the highest levels of salinity, the microbial community tends to have a considerable similarity with control samples of non-saline soils.

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As rochas evaporíticas se destacam nos estudos sedimentológicos e estratigráficos por sua associação com rochas geradoras de petróleo como rochas selantes e por seu significado paleoclimático e paleoambiental. A caracterização faciológica e petrográfica quantitativa permitiu a definição das fácies e hábitos dos evaporitos do Membro Ipubi da Bacia do Araripe, coletados em afloramentos localizados principalmente na região de Araripina, Pernambuco. Técnicas de difratometria de raios X (DRX) e microscopia eletrônica de varredura (MEV) foram utilizadas com o intuito de complementar a análise dos aspectos composicionais e texturais dos constituintes. Os sulfatos dominantes são gipsita e anidrita, localmente celestita. Foram identificadas macroscopicamente três fácies (Evaporito laminado, Evaporito maciço e Evaporito fraturado) e microscopicamente treze hábitos para os sulfatos. Dentre estes, os hábitos e as texturas primários são anidrita nodular, gipsita paliçada e chevron, estas duas últimas constituindo a textura laminada. Os demais sulfatos foram precipitados em condições pós-deposicionais rasas. As análises isotópicas de S e O nos sulfatos forneceram valores de δ34S entre +10,27‰ e +17,99‰ e δ18O entre +7,72‰ e +13,30‰, caracterizando a composição marinha da salmoura geradora dos depósitos. Os resultados obtidos indicam que os evaporitos do Membro Ipubi foram depositados em contexto subaquoso (salinas) e intrasedimentar em um sabkha costeiro.

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Resumo - O Stock Mingu é um granito do Domínio Macururé, Sistema Orogênico Sergipano (SOS), sul da Província da Borborema. As suas mineralizações de fluorita e sulfetos de Cu e Pb foram geradas por processos hidrotermais, evidenciados pelos filões mineralizados e pela cor avermelhada das rochas graníticas que os contém. As rochas do stock são biotita monzogranitos, quartzo monzonitos e monzogranitos, os dois últimos encaixantes dos filões. A química destas rochas evidenciou afinidade com a série shoshonítica. No contacto com estes granitos encontra-se a Unidade Batalha constituída por metadolomitos. A mineralogia dos filões é constituída por quartzo 1 e 2, fluorita, calcita, calcopirita, galena e variados carbonatos, óxidos e sulfatos. O estudo de inclusões fluidas revelou que os fluidos circularam a temperaturas no intervalo de 350 °C a 94 °C, ocorrendo as principais fases de deposição dos minerais dos filões em média a 170 °C e 120 °C. São fluidos salinos com salinidades médias entre 5 e 15 wt% NaCl eq., e as temperaturas dos eutéticos indicam provável presença dos cátions bivalentes K, Ca, Mg. Estes fluidos, resultando provavelmente da influência dos metadolomitos da Unidade Batalha sobre fluídos que circulavam pela falha Belo Monte-Jeremoabo, foram os responsáveis pelos processos hidrotermais no Stock Mingu. Palavras-chave: Mineralizações hidrotermais de fluorita. Inclusões fluidas. Sul da Província Borborema. Sistema Orogênico Sergipano. Abstract - The Mingu Stock is a granite from the Macururé Domain, Sergipano Orogenic System (SOS), south of Borborema Province. Its fluorite and Cu and Pb sulphide mineralizations were generated by hydrothermal processes, evidenced by the mineralized veins and by the reddish color of the granitic rocks that contain them. The rocks in the stock are biotite monzogranites, quartzo monzonites and monzogranites, the last two host veins. The chemistry of these rocks showed affinity with the shoshonitic series. In contact with these granites is the Batalha Unit made up of metadolomites. The mineralogy of the veins consists of quartz 1 and 2, fluorite, calcite, chalcopyrite, galena and various carbonates, oxides and sulfates. The study of fluid inclusions revealed that the fluids circulated at temperatures ranging from 350 °C to 94 °C, with the main phases of deposition of the vein minerals occurring on average at 170 °C and 120 °C. They are saline fluids with average salinities between 5 and 15 wt% NaCl eq., and the temperatures of the eutectics indicate the probable presence of the bivalent cations K, Ca, Mg. These fluids, probably resulting from the influence of the Batalha Unit's metadolomites on fluids that circulated through the Belo Monte-Jeremoabo fault, were responsible for the hydrothermal processes in Stock Mingu. Keywords: Hydrothermal Fluorite Mineralizations. Fluid Inclusions. Southern Borborema Province. Sergipe Orogenic System.

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Objective Over the past several years, only approximately 50% of HIV-exposed infants received an early infant diagnosis test within the first two months of life. While high attrition and mortality account for some of the shortcomings in identifying HIV-infected infants early and putting them on life-saving treatment, fragmented and challenging laboratory systems are an added barrier. We sought to determine the accuracy of using HIV viral load assays for infant diagnosis of HIV. Methods We enrolled 866 Ugandan infants between March–April 2018 for this study after initial laboratory diagnosis. The median age was seven months, while 33% of infants were less than three months of age. Study testing was done using either the Roche or Abbott molecular technologies at the Central Public Health Laboratory. Dried blood spot samples were prepared according to manufacturer-recommended protocols for both the qualitative and quantitative assays. Viral load test samples for the Roche assay were processed using two different buffers: phosphate-buffered saline (PBS: free virus elution viral load protocol [FVE]) and Sample Pre-Extraction Reagent (SPEX: qualitative buffer). Dried blood spot samples were processed for both assays on the Abbott using the manufacturer’s standard infant diagnosis protocol. All infants received a qualitative test for clinical management and additional paired quantitative tests. Results 858 infants were included in the analysis, of which 50% were female. Over 75% of mothers received antiretroviral therapy, while approximately 65% of infants received infant prophylaxis. The Roche SPEX and Abbott technologies had high sensitivity (>95%) and specificity (>98%). The Roche FVE had lower sensitivity (85%) and viral load values. Conclusions To simplify and streamline laboratory practices, HIV viral load may be used to diagnose HIV infection in infants, particularly using the Roche SPEX and Abbott technologies.

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Background:Fenebrutinib (GDC-0853, FEN) is an oral, non-covalent, and selective inhibitor of Bruton’s tyrosine kinase (BTK) in clinical development for autoimmune diseases.Objectives:This was a randomized, placebo-controlled, multi-center study to evaluate the efficacy, safety, and pharmacodynamic effects of FEN in patients with moderate-to-severe systemic lupus erythematosus (SLE) activity.Methods:Patients who met SLICC or revised ACR SLE criteria, had ≥1 serologic marker of SLE, SLEDAI ≥8, and were on ≥1 standard of care (SOC) therapy were included; patients with renal or CNS involvement, or exposure to B cell depleting or calcineurin inhibitor therapy were excluded. Patients were randomized to placebo (PBO), FEN 150 mg QD, or FEN 200 mg BID, for 48 weeks. A corticosteroid taper was recommended, with burst and taper permitted from Week 0 (W0) to W12 and W24 to W36. The primary endpoint was SRI-4 at W48. Post hoc subgroup analyses were conducted based on patient baseline disease characteristics.Results:This study enrolled 260 patients, with the majority recruited in Latin America, USA, and Western Europe. At W48, the SRI-4 response rates for FEN 150 mg QD and FEN 200 mg BID were 51% (95% CI: -8.5, 21.2; p value 0.37) and 52% (95% CI: -7.3, 22.4; p value 0.34), respectively, compared to 44% for PBO (Table 1). Post-hoc analysis showed larger responses in subgroups of patients with higher baseline disease activity (Table 1). Safety results were similar between FEN and PBO arms, although more serious adverse events were observed in the FEN 200 mg BID arm. Study discontinuations were balanced across the 3 arms (24-26%). FEN treatment significantly reduced levels of CD19+ B cells, anti-dsDNA autoantibodies, IgG, and a BTK-dependent RNA signature highly expressed in plasmablasts by W48 compared to PBO; C4 levels modestly improved with FEN vs. PBO (Table 2).Table 1.SRI-4 Response (%) at W48 in Primary Analysis and in Post-hoc Patient SubgroupsPBOFEN 150 mg QDFEN 200 mg BIDSRI-4 Response (%) at W4844n=8451n=8752n=88SRI-4 Response (%) in Baseline Subgroups At least 1 BILAG A48n=4254n=3959n=46 At least 1 BILAG A and SLEDAI increased DNA binding37n=1953n=1765n=26 SLEDAI arthritis with at least 4 swollen joints39n=5750n=5457n=54 SLEDAI arthritis with at least 4 tender joints39n=7153n=7059n=69 CLASI >=1021n=1436n=1131n=16Table 2.Key Biomarker ResultsPBOFEN 150 mg QDFEN 200 mg BIDMedian (%) Change from Baseline at W48 Plasmablast signature-19.7%n=52-54.3%*n=53-51.7%*n=57 CD19+B cells (cells/µl)-0.50n=38-57.0*n=49-57.5*n=48 Anti-dsDNA#(IU/ml)+6.9n=31-38.3*n=36-75.7*n=33 Total IgG (g/L)-0.20n=65-1.25*n=64-1.56*n=64 C3 (g/L)-0.02n=65+0.01n=67-0.01n=66 C4 (g/L)0.00n=65+0.02*n=67+0.01*n=66#Patients who were positive at baseline (>30 IU/mL)*Denotes significant vs. PBO; Kruskal-Wallis false-discovery rate controlled two sided (p-value ≤0.05)Conclusion:The primary endpoint of SRI-4 for FEN was not met despite evidence of strong BTK target and pathway inhibition. FEN had an acceptable safety profile. Several disease activity subgroups were suggestive of a greater treatment effect on SRI-4 compared to PBODisclosure of Interests:David Isenberg Consultant of: Study Investigator and Consultant to Genentech, Richard Furie Grant/research support from: AstraZeneca, Biogen, Consultant of: AstraZeneca, Biogen, Nicholas S. Jones Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Pascal Guibord Shareholder of: Roche, Employee of: Roche, Joshua Galanter Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Chin Lee Shareholder of: Genentech/Roche and Eli Lilly, Employee of: Genentech/Roche, Anna McGregor Employee of: Genentech/Roche, Balazs Toth Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Julie Rae Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Olivia Hwang Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Armend Lokku Shareholder of: Roche, Employee of: Roche, Pedro Miranda Consultant of: Study Investigator for Genentech, Viviane de Souza Consultant of: Study investigator for Genentech, Juan Jaller-Raad Consultant of: Study investigator for Genentech, Anna Maura Fernandes Consultant of: Study investigator for Genentech, Rodrigo Garcia Salinas Consultant of: Study investigator for Genentech, Leslie Chinn Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Michael J. Townsend Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Alyssa Morimoto Shareholder of: Genentech/Roche, Employee of: Genentech/Roche, Katie Tuckwell Shareholder of: Genentech/Roche, Employee of: Genentech/Roche

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ABSTRACT Although Roche COBAS Ampliprep/COBAS TaqMan (CAP/CTM) systems are widely used in sub-Saharan Africa for early infant diagnosis of HIV from dried blood spots (DBS), viral load monitoring with this system is not practical due to nonspecific extraction of both cell-free and cell-associated viral nucleic acids. A simplified DBS extraction technique for cell-free virus elution using phosphate-buffered saline (PBS) may provide an alternative analyte for lower-cost quantitative HIV virus load (VL) testing to monitor antiretroviral therapy (ART). We evaluated the CAP/CTM v2.0 assay in 272 paired plasma and DBS specimens using the cell-free virus elution method and determined the level of agreement, sensitivity, and specificity at thresholds of target not detected (TND), target below the limit of quantification (BLQ) (<20 copies/ml in plasma or <400 copies/ml in DBS), and VL of <1,000 copies/ml, and VL of <5,000 copies/ml. Reported plasma VL ranged from TND, or <20, to 5,781,592 copies/ml, and DBS VL ranged from TND, or <400, to 467,600 copies/ml. At <1000 copies/ml, agreement between DBS and plasma was 96.7% (kappa coefficient, 0.93; P < 0.0001). The mean difference between DBS and plasma VL values was −1.06 log 10 copies/ml (95% confidence interval [CI], −1.17, −0.97; P < 0.0001). At a treatment failure threshold of >1,000 copies/ml, the sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) were 92.7%, 100%, 100%, and 94.3%, respectively. PBS elution of DBS offers a sensitive and specific method for monitoring plasma viremia among adults and children on ART at the WHO-recommended threshold of >1,000 copies/ml on the Roche CAP/CTM system.

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Introduction: Trauma-induced coagulopathy (TIC) is a multifactorial condition secondary to severe trauma. In TIC, early fibrinogen (FI) replacement and low dose of recombinant activated factor VII (rFVIIa) may positively impact outcome. Factor XIII (FXIII), on the other hand, may stimulate in vitro clot formation and clot stability. We hypothesized that combination of FI, rFVIIa and FXIII might normalize clot formation more effectively than the isolated use of each concentrate in a model of TIC. Aim: Evaluation of the procoagulant effect of isolated or combined use of FI, rFVIIa and FXIII in a model of TIC. Methods: TIC in vitro model was obtained by dilution of whole blood from seven healthy controls with isotonic saline (NaCl 0.9%) (2:3 whole blood:saline ratio). FI, rFVIIa and FXIII were spiked in combination or alone until obtaining final levels of 2 g/L, 1 μg/mL and 100 IU/dL respectively. Procoagulant effects of the different concentrates or their mixtures were evaluated by Rotational Thromboelastometry (ROTEM®, Werfen) triggered using starTEM® (calcium chloride 0,2 M) and exTEM® reagent (source of tissue factor) diluted with saline up to 1:100.000 (final dilution) for a better evaluation of both the extrinsic and intrinsic pathways of coagulation. The values of clotting time (CT: time until 2 mm of amplitude, in seconds), amplitude (parameter proportional to the clot strength) at 5 minutes (A5, in mm) and clot formation time (CFT: time from CT to 20 mm of amplitude, in seconds) were evaluated. Statistical analysis of differences was performed by One-Way ANOVA test assuming no paring of data and using the Holm-Sidak's correction for multiple comparisons with a family-wise significance and confidence level of 0.01. Statistical significance was set at p&lt; 0.05. Results/Discussion: Data are summarized in Table I and Figure 1. CT needed the combination of two of more concentrates to reach the normal range suggesting that the administration of FI alone in TIC may not be enough to restore the patients' hemostatic potential. In regard to the clot strength evaluated by A5, the addition of FXIII or rFVIIa alone or in combination did not improve the value of A5 that was only normalized by the addition of FI. This effect of FI was increased in the presence of FXIII or rFVIIa which indicated that normal levels of FI might be required for rFVIIa or FXIII to be effective emphasising the possible benefit of the combinatory therapy. Like observed in A5, the velocity of clot formation evaluated by the CFT was normalised only by the addition of FI. However, the combination of FI plus FXIII + rFVIIa had a stronger effect on CFT compared with the combination of FI + FXIII or FI + rFVIIa, indicating that the improvement of thrombin generation due to rFVIIa plus an increment of fibrin formation and net stabilization through the contribution of higher levels of FI and FXIII respectively, might provide a beneficial synergistic procoagulant effect in TIC. Conclusion: The use of FI in TIC may contribute to increase the patient's hemostatic potential but might not be enough. Combinatory therapies based on the administration of FI, rFVIIa and FXIII might be of better benefit in this setting. Ex-vivo studies using blood of patients with stablished TIC might bring new insights on the possible advantages of this combinatory therapy to design more effective protocols to treat this frequent and life-threatening acquired condition. Disclosures Canales: Sandoz: Honoraria; iQone: Honoraria; Janssen: Speakers Bureau; Janssen: Honoraria; Roche: Speakers Bureau; Karyopharm: Honoraria; Sandoz: Speakers Bureau; Novartis: Honoraria; Takeda: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Janssen: Speakers Bureau; Roche: Speakers Bureau; Sandoz: Speakers Bureau; Takeda: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Gilead: Honoraria. Butta:NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Pfizer: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Grifols: Research Funding. Alvarez Román:NovoNordisk,: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy; Grifols: Research Funding. Jiménez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria.

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Abstract Background Serum free light chain (FLC) assays are used clinically to measure the concentration of κ and λ FLC in patients with suspected or diagnosed plasma cell proliferative disorders. Previous studies have demonstrated a loss of linearity in low concentration ranges of these assays. We hypothesized that this result could be caused by a matrix effect. Methods Recovery studies were performed for κ and λ FLC in both serum and saline using the Freelite assay (Binding Site) on a Cobas c502 system (Roche). Samples were analyzed either at the recommended dilution or undiluted. Follow-up studies were performed in varying matrices ranging from 0% to 100% saline. Retrospective patient data were analyzed to assess the impact on reported κ FLC, λ FLC, and κ/λ ratio. Results FLC in a serum matrix demonstrated underrecovery relative to samples diluted in saline for both κ and λ FLC. Of 255 patient samples with λ FLC measured undiluted (λ FLC &lt;6.0 mg/L), an unexpected gap was observed in patient results between 2.0 and 6.0 mg/L. In addition, 23 patients measured serially with λ FLC between 2.0 and 6.0 mg/L demonstrated dramatic changes in κ/λ ratio, with no changes in κ FLC, likely because of the matrix effect. Conclusions The κ and λ Freelite assays exhibit a matrix effect when samples are tested undiluted, which has the potential to affect the κ/λ ratio. Consequently, our laboratory has stopped reporting λ FLC &lt;6.0 mg/L.

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Abstract Abstract 4920 Within a prospective phase II study (HOVON 80) of patients with recurrent diffuse large B-cel lymphoma in the CNS, three of 13 patients treated with intrathecal rituximab developed an acute, transient, extremely painful lumbosacral radiculopathy. All were treated with systemic R-DHAP every 4 weeks with i.v. HD-MTX on day 15 for three cycles. In addition intrathecal Rituximab was administered twice weekly via lumbar puncture starting on day -1. According to protocol the first administration consisted of 10 mg of rituximab after premedication with acetaminophen, thereafter the dose was increased to 25 mg. No patient experienced side effects of the first intrathecal administration of rituximab. However, after the first administration of 25 mg rituximab three of 13 treated patients reported extremely painful tingling sensations in the buttocks, legs and feet immediately after administration and lasting 30–60 minutes. Concomitantly a temporarily increased bloodpressure was documented. Premedication with an antihistaminic in the third patient was ineffective. No neurologic deficits occurred and the pain resolved completely. The patients refused further treatment with intrathecal rituximab, and therapy was changed to intrathecal methotrexate, without any side effects. After these events the rituximab was diluted in saline to 5 mg/ml, the dose reduced to 10 mg per administration, and 4 mg dexamethasone was administered concomitantly in all subsequent patients. Twelve additional patients were thus treated and no further incidents of painful radiculopathy were observed. This serious, though completely transient, adverse effect of intrathecal rituximab precludes intrathecal administration of higher doses of rituximab via lumbar route. It has never been described after intraventricular administration. Disclosures: Bromberg: Roche: Research Funding. Off Label Use: rituximab administration intrathecally. Doorduyn: Roche: Research Funding. van den Bent: Roche: Consultancy, Research Funding.

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Na região norte do estado do Piauí, nordeste do Brasil, a perfuração de poços tem sido uma alternativa ao precário sistema de abastecimento público, no entanto, a concentração de STD na água pode limitar seu uso para abastecimento. O objetivo deste trabalho foi realizar uma análise espacial da concentração de sólidos totais dissolvidos (STD) em águas subterrâneas da região norte do Piauí. Para tanto, realizou-se uma interpolação dos valores de STD registrados nos poços da região, com uso de método geoestatístico. Posteriormente, buscou-se correlacioná-los com as características geológicas da região. O resultado demonstrou que as águas doces ocorrem em uma área de 201 km², enquanto que as águas salobras e salinas ocorrem em uma área 796 e 1.180 km², respectivamente e apresentaram correspondência espacial com as áreas de afloramento de rochas da suíte intrusiva Chaval. As áreas que apresentaram maior estimativa de ocorrência de água doce estão localizadas nas proximidades da foz dos rios Camurupim e Igaraçu, e nas demais porções da faixa litorânea a estimação da ocorrência de STD aponta para uma salinização das águas subterrâneas, que possivelmente pode estar associado a intrusão salina devido a exploração excessiva do manancial subterrâneo.Na região norte do estado do Piauí, nordeste do Brasil, a perfuração de poços tem sido uma alternativa ao precário sistema de abastecimento público, no entanto, a concentração de STD na água pode limitar seu uso para abastecimento. O objetivo deste trabalho foi realizar uma análise espacial da concentração de sólidos totais dissolvidos (STD) em águas subterrâneas da região norte do Piauí. Para tanto, realizou-se uma interpolação dos valores de STD registrados nos poços da região, com uso de método geoestatístico. Posteriormente, buscou-se correlacioná-los com as características geológicas da região. O resultado demonstrou que as águas doces ocorrem em uma área de 201 km², enquanto que as águas salobras e salinas ocorrem em uma área 796 e 1.180 km², respectivamente e apresentaram correspondência espacial com as áreas de afloramento de rochas da suíte intrusiva Chaval. As áreas que apresentaram maior estimativa de ocorrência de água doce estão localizadas nas proximidades da foz dos rios Camurupim e Igaraçu, e nas demais porções da faixa litorânea a estimação da ocorrência de STD aponta para uma salinização das águas subterrâneas, que possivelmente pode estar associado a intrusão salina devido a exploração excessiva do manancial subterrâneo.

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Studies agree that searchers are often not satisfied with the performance of current enterprise search engines. As a consequence, more scientists worldwide are actively investigating new avenues for searching to improve retrieval performance. This paper contributes to YASA (Your Adaptive Search Agent), a fully implemented and thoroughly evaluated ontology-based information retrieval system for the enterprise. A salient particularity of YASA is that large parts of the ontology are automatically filled with facts by recycling and transforming existing data. YASA offers context-based personalization, faceted navigation, as well as semantic search capabilities. YASA has been deployed and evaluated in the pharmaceutical research department of Roche, Penzberg, and results show that already semantically simple ontologies suffice to considerably improve search performance.

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AbstractWe report a microfluidic sandwich immunoassay constructed around a dual-giant magnetoresistance (GMR) sensor array to quantify the heart failure biomarker NT-proBNP in human plasma at the clinically relevant concentration levels between 15 pg/mL and 40 ng/mL. The broad dynamic range was achieved by differential coating of two identical GMR sensors operated in tandem, and combining two standard curves. The detection limit was determined as 5 pg/mL. The assay, involving 53 plasma samples from patients with different cardiovascular diseases, was validated against the Roche Cobas e411 analyzer. The salient features of this system are its wide concentration range, low detection limit, small sample volume requirement (50 μL), and the need for a short measurement time of 15 min, making it a prospective candidate for practical use in point of care analysis.

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The aim of our study was to compare the diagnostic performance of cardiac troponin T (cTnT) and cardiac troponin I (cTnI) in three groups of rabbits: 1) control (saline 1 ml/kg i.v.); 2) daunorubicin (3 mg/kg i.v.); 3) daunorubicin (3 mg/kg i.v.) + dexrazoxane (60 mg/kg i.p.). The drugs were given once a week, 10 administrations. The concentration of cTnT was measured using Elecsys Troponin T STAT Immunoassay (Roche). The concentration of cTnI was measured using AxSYM Troponin I (Abbott). The linear regression model was applied to see if there is a dependence between cTnT and cTnI. The coefficient of determination (R2 = 0.79) was acceptable only in the control group. In the remaining cases (i.e. in the daunorubicin group and in the daunorubicin + dexrazoxane treated group) R2 was too small (0.53, and 0.06). We may conclude that in rabbits after repeated administration of cardiotoxic or cardioprotective drugs meaningful dependence between cTnT and cTnI was not found. The choice of the most suitable cardiomarker in laboratory animals deserves further studies.

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O trabalho compõe-se de uma lista de briófitas, um grupo de plantas ausente em ambientes marinhos, porém ocorrente em condições arenosas e salinas e sob ação das ondas e dos ventos nas praias de Ubatuba, Bertioga e Peruíbe, no Estado de São Paulo. O material coletado encontra-se depositado nos herbários SP e HRCB. As 108 exsicatas com 67 amostras de hepáticas e 59 amostras de musgos incluem 25 famílias, 49 gêneros e 77 espécies de briófitas. Os maiores números de gêneros e espécies foram observados em Lejeuneaceae e Lejeunea, respectivamente, entre as hepáticas. O mesmo pode ser dito para Orthotrichaceae e Bryum, quanto aos musgos. São citadas 29 espécies, pela primeira vez, para determinados tipos de substratos como solo, casca de arbustos e rochas. As hepáticas Ceratolejeunea laetefusca (Austin) R.M. Schust., Colura ulei Ast., Lejeunea bermudiana (A. Evans) R.M. Schust. e os musgos Calymperes afzelii Sw., Fissidens serratus Müll. Hal. e Weissia controversa Hedw. são citados pela primeria vez para o Estado de São Paulo. As hepáticas superaram os musgos em número de gêneros e espécies. Todas as espécies listadas são citadas pela primeira vez para praias.

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Abstract Background Widespread testing of SARS-CoV-2 has resulted in shortages of collection devices and transport media. We evaluated the stability of flocked swabs inoculated with SARS-CoV-2-containing specimen incubated dry (i.e., without transport medium) at room temperature. Methods A pool of SARS-CoV-2 positive specimen was used to inoculate flocked swabs. Five swabs were placed immediately into universal transport media (UTM) following inoculation, and tested immediately (day 0). Fifteen of the swabs were placed into sterile 15-mL conical tubes and incubated at room temperature for 1, 2, or 7 days. Following incubation, swabs were hydrated in separate vials of UTM and tested. This protocol was repeated for viral transport media (VTM) and saline. As a comparison, a series of swabs was prepared and tested in parallel, but stored in the corresponding liquid transport media (UTM, VTM, or saline) and incubated at room temperature. Testing was performed at 1, 2, and 7 days postinoculation in duplicate. All molecular testing was performed using the Roche cobas SARS-CoV-2 assay. Results All dry swabs tested on days 1, 2, and 7 provided results that were within 2 cycle thresholds (CTs) of the average CT values for swabs hydrated in the same media and tested on day 0. There was no statistical difference in CT values between swabs incubated in liquid media versus dry swabs incubated at room temperature prior to hydration in liquid media. Conclusions The utilization of “dry swabs” may simplify specimen collection, negate the need for liquid transport media, and mitigate safety risks while preserving the accuracy of testing.

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Both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are considered to be reliable biomarkers with sufficient sensitivity and specificity for cardiac injury in the majority of laboratory animals. The aim of our study was to compare the diagnostic performance of cTnT and cTnI in three groups of rabbits: 1) control (saline 1 ml/kg i.v.); 2) Salicylaldehyd Isonicotinoyl Hydrazone – SIH (50 mg/kg, once weekly, i.p.; partially dissolved in 10 % Cremophor solution); 3) 10 % Cremophor solution in water (2 ml/kg i.v.). The drugs were given once a week, 10 administrations. The concentration of cTnT was measured using Elecsys Troponin T STAT Immunoassay (Roche). The concentration of cTnI was measured using AxSYM Troponin I (Abbott). The linear regression model was applied to see if there is a dependence between cTnT and cTnI. The coefficient of determination was not acceptable in all groups. The highest value of R2was found in the control group (R2= 0.424). We may conclude that in rabbits meaningful dependence between cTnT and cTnI was not found. According to our long-term experiences cTnT seems to be more suitable cardiomarker in rabbits in comparison with cTnI where the data are characterized by the large scatter.

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The authors examined the effect of selective endothelin (ET) receptor type A (ETA) antagonism on histological and functional recovery in cat at 24 hours after reversible middle cerebral artery occlusion (MCAO). A novel and specific ETA antagonist, Ro 61-1790 [5-methylpyridine-2-sulfonic acid-6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(2-1 H-tetrazol-5-yl-pyridin-4-yl)-pyrimidin-4-ylamide sodium salt (1:2)] (Roche, Basel, Switzerland), was used at doses that produced steady-state plasma concentrations and abolished ET-induced pial arteriolar vasoconstriction. In a cranial window preparation, 8 nmol/L ET constricted pial arterioles by 33 ± 18% (mean ± SD), but this response was ablated by intravenous Ro 61-1790 treatment (10-mg/kg bolus, 4-mg/kg/h infusion). In additional animal cohorts, halothane-anesthetized cats were treated with 90 minutes of MCAO and 24 hours of reperfusion. Animals received Ro 61-1790 infusion beginning at the onset of reperfusion and continuing for 6 or 24 hours (n = 41). Control cats were treated with 0.9% saline by intravenous infusion throughout reperfusion. There was no difference in injury volume or neurologic evaluation score in saline-treated cats (n = 11; caudate 24 ± 28%, cortical injury 7.5 ± 5% of ipsilateral structure; score 52 ± 8) versus the results in cats treated with Ro 61-1790 for either 24 hours (n = 6; caudate 22 ± 23%, cortex 6 ± 5%, injury volume of ipsilateral structure; score 55 ± 3) or 6 hours (n = 11; caudate 33 ± 30%, cortex 12 ± 14%, injury volume of ipsilateral structure; score 50 ± 10). Mortality was greatest in the 24-hour drug treatment group. These data suggest that blockade of ETA receptor activity is not beneficial to tissue or functional outcomes from experimental stroke in cat.

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N-Chlorotaurine (NCT) is a promising endogenous agent for topical treatment of infections. We tested the tolerability and pharmakokinetics of NCT in the bovine mammary glands in a phase 1 study. Three concentrations of NCT in water (0·1%, 1·0%, 2·0%) were administered intramammarily in each of two cows. Into two quarters of the udder 100 ml NCT was injected into each twice daily for 5 d, while 0·9% NaCl was injected into the other two quarters in a randomized and blinded manner. Samples of milk were taken to determine the number of leucocytes and the activity of NCT, and samples of urine and blood to determine the taurine and chloride concentration. Chloride concentrations in serum samples were determined by an ISE-Unit of a Modular-System of the Roche Diagnostics company. The udder was monitored clinically for signs of inflammation. Oxidative activity could be detected in the milk after single irrigations for 15 min (0·1% NCT) and for maximally 5 h (1% and 2% NCT), respectively. On day 2, leucocytes increased to 4×106/ml in the NCT group, while they remained ⩽1×106/ml in the saline group. However, on days 3–5 they increased to (5–7)×106 in both the NCT and control group without any statistical difference. One day after the end of dosing the number decreased significantly and reached the baseline (<1×106/ml) on day 10. The decrease was similar in both groups. Except for sporadic slight induration of single quarters in both groups and slight reduction of milk performance no disorders occurred. Taurine levels in blood and urine did not change. Irrigation of the bovine mammary gland with both NCT and saline caused a transient increase of leucocytes in the milk, but no severe side effects. The absence of residues and decay products may be a great advantage of NCT over other antimicrobial agents.

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Background:Intra-articular therapies (IAT) are routinely used in rheumatic and musculoskeletal diseases (RMDs); however large variability exists regarding current practice of delivery amongst health professionals.Objectives:To inquire about common practice aspects to inform the EULAR Taskforce for the IAT of arthropathies.Methods:A steering committee prepared a 160-item questionnaire based on the information needs of the Taskforce. The survey was disseminated via EULAR professional associations and social media and it was open to any health professional treating persons with RMDs, regardless of using IAT personally.Results:The survey was answered by 186 health professionals from 26 countries, the large majority of whom (77%) were rheumatologists, followed by nurses (12%), general practitioners (2%) and orthopaedic surgeons (2%). The two collectives that perform IAT routinely are rheumatologists (97%) and orthopaedic surgeons (89%), with other professionals <50%. Specific training was compulsory for 32%. The most frequent indication for IAT is inflammatory arthritis (76%), followed by osteoarthritis (74%), crystal arthritis (71%) and bursitis (70%); and all joints are injected, with knee (78%) and shoulder (70%) being the most frequent. When questioned about specific contexts, such as pre-surgical, diabetic or hypertensive patients, variability among respondents was evident, with around 30 to 69% of professionals considering it acceptable to inject glucocorticoids (GC), while in others there was less variability (prosthetic or septic joints, <1%). GCs are the most used compounds, followed by hyaluronic acid and saline/dry puncture. Only 66 (36%) use ultrasound to guide IAT. In their opinion, to be accurately in the joint is moderately to largely important for large joints (80%) and very important in small joints. The maximum number of injections to perform safely in the same joint within one year was “2 to 3” for 65% (2% thought there is “No limit”). The majority reported that they informed patients about side-effects (73%), benefits (72%), and the nature of the procedure (72%), and less frequently about other aspects; with 10% obtaining written consent and 56% oral consent (mandatory only for 32%). Other questions help to understand the setting and procedures followed, including use of local anaesthetics and care after injection.Conclusion:Although often performed in clinical practice for RMDs, there is apparent variability in several elements related to delivery of this treatment. This information, together with patient input, will help design current recommendations where research evidence is not available.Acknowledgments:Eular Taskforce grant CL109Disclosure of Interests:Jenny de la Torre-Aboki: None declared, IRENE Pitsillidou: None declared, Jacqueline Uson Jaeger: None declared, Esperanza Naredo: None declared, Lene Terslev: None declared, Mikael Boesen Consultant of: AbbVie, AstraZeneca, Eli Lilly, Esaote, Glenmark, Novartis, Pfizer, UCB, Paid instructor for: IAG, Image Analysis Group, AbbVie, Eli Lilly, AstraZeneca, esaote, Glenmark, Novartis, Pfizer, UCB (scientific advisor)., Speakers bureau: Eli Lilly, Esaote, Novartis, Pfizer, UCB, Hemant Pandit Grant/research support from: Glaxo Smith Kline (GSK) for work on Diclofenac Gel, Speakers bureau: Bristol Myers Squibb for teaching their employees about hip and knee replacement, Ingrid Möller: None declared, Maria Antonietta D’Agostino Consultant of: AbbVie, BMS, Novartis, and Roche, Speakers bureau: AbbVie, BMS, Novartis, and Roche, Willm Uwe Kampen: None declared, Terence O’Neill: None declared, Michael Doherty Grant/research support from: AstraZeneca funded the Nottingham Sons of Gout study, Consultant of: Advisory borads on gout for Grunenthal and Mallinckrodt, Francis Berenbaum Grant/research support from: TRB Chemedica (through institution), MSD (through institution), Pfizer (through institution), Consultant of: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Bone Therapeutics, Regulaxis, Peptinov, 4P Pharma, Paid instructor for: Sandoz, Speakers bureau: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Sandoz, Valentina Vardanyan: None declared, Elena Nikiphorou: None declared, Sebastian C Rodriguez-García Speakers bureau: Novartis Farmaceutica, S.A., Merck Sharp & Dohme España, S.A., Sanofi Aventis, UCB Pharma, Raul Castellanos-Moreira: None declared, Loreto Carmona Grant/research support from: Novartis Farmaceutica, SA, Pfizer, S.L.U., Merck Sharp & Dohme España, S.A., Roche Farma, S.A, Sanofi Aventis, AbbVie Spain, S.L.U., and Laboratorios Gebro Pharma, SA (All trhough institution)

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A análise de aspectos fisiográficos, bióticos e antrópicos das bacias hidrográficas deve oferecer subsídios efetivos para o processo de determinação de pontos de maiores e menores restrições hídricas, limites de possíveis irreversibilidades e indicação regionalizada de alternativas de manejo. Nesse contexto, é preciso desenvolver e testar instrumentos destinados à identificação da interação entre o processo de apropriação humana do território e a base natural, em compartimentos espaciais internos às bacias. O presente texto se atém aos aspectos naturais, procurando avaliar a possibilidade de aplicação da abordagem paisagística à gestão de recursos hídricos . A parte mineira da bacia do rio Jequitinhonha foi tomada como estudo de caso. Verifica-se que a região mais heterogênea da bacia abrange a margem esquerda do rio Jequitinhonha, das cabeceiras até a sub-bacia do rio Salinas, onde ocorrem todos os tipos de litologia e feições morfológicas, vegetação e solos presentes na totalidade da área de estudo. Na margem direita do mesmo rio, incorporando a quase totalidade da sub-bacia do rio Araçuaí, predominam amplas chapadas capeadas por sedimentos cenozóicos, vegetação de cerrado e manchas de floresta estacional. No Médio Jequitinhonha, estendendo-se do município de Araçuaí até o extremo jusante da área de estudo, em ambas as margens, predominam as rochas graníticas e gnáissicas, maciços estruturais e intrusões em forma de pontões, vegetação de caatinga no setor oeste e florestas estacionais no setor leste. O relevo fortemente ondulado e os solos muito susceptíveis à erosão são características presentes em todas as unidades de paisagem.

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Context.—DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. Objective.—To demonstrate the fundamental principles of pyrosequencing. Data Sources.—Salient features of pyrosequencing are demonstrated using the free software program Pyromaker (http://pyromaker.pathology.jhmi.edu), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. Conclusions.—We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.

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Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptotic bax, two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia–reperfusion, and to the combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia–reperfusion. Brain hom*ogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; “chemical preconditioning”) or vehicle (normal saline), and were either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro-and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro-and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical preconditioning using 3-NPA in rats.

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Los dinteles y los escalones labrados del Edificio 33 de la Zona Arqueológica de Yaxchilán, Chiapas, México, han presentado, desde que inició su monitoreo sistemático por parte del área de conservación del inah, en 1989, alteraciones propias de una roca caliza en proceso de intemperismo prolongado. Sin embargo, en 2003 se hizo patente que estos elementos se estaban alterando de manera distinta de la observada hasta ese momento, ya que súbitamente sus superficies presentaban desprendimientos en forma de escamas, halos y velos blanquecinos localizados, manchas de colores y material pulverulento que no habían sido apreciados con anterioridad. ____ Since the conservation department of inah started the systematic monitoring of the carved stairs and lintels of Structure 33 at the archaeological site of Yaxchilán, Chiapas, México, in 1989, these elements have developed weathering alterations in the limestone rock. However, by 2003 conservators noticed specific and accelerated effects of decay, such as flaking, saline veils and halos, coloured stains and dusty material, which had not been hitherto reported. This research exposes a methodological analysis of the probable causes and mechanisms of alteration of these phenomena; it also poses various lines of investigation in order to promote the preservation of these significant Classic Mayan sculptures.

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BackgroundSystemic autoimmunity is a complex and multifactorial process for which underlying mechanism are unclear. Several theories have been formulated to explain the development of autoimmunity, including excessive self-immunosurveillance, danger, and antigen discontinuity. The antigen discontinuity and the self-criticality theories presume that repetitive antigen exposure leads to a dysregulated immune response. Supporting these theories, Tsumiyama and Shiozawa have shown that repetitive immunization of BALB/c mice with ovalbumin induces auto-antibodies production and a glomerulonephritis with immune deposit resembling lupus nephritis [1].These data prompted us to consider the potential impact of repetitive SARS-CoV-2 infection in the onset of autoimmune diseases in human. Indeed, since 2020, a large majority of the population has encountered SARS-CoV-2 antigens multiples times, either through vaccination or through infection/reinfection. Hence, we hypothesized that repetitive exposure to the SARS-CoV-2 spike antigen may induce autoimmunity in healthy individuals.ObjectivesEvaluate whether repetitive exposure to SARS-CoV-2 spike protein induces autoimmunity in mice.MethodsWe immunized intraperitoneally C57Bl/6 mice (n = 5/group) with recombinant SARS-CoV-2 spike protein (1µg per injection) or vehicle (phosphate buffered saline [PBS]);Figure 1A). The first injection was mixed with Alum as an adjuvant, and the other injections were diluted in PBS, administered intraperitoneally every 5 days, 16 times in total. Five days after the final injection, the mice were sacrificed to retrieve serum. We measured anti-spike and anti-dsDNA IgG using ELISA. The serum from 5 34-week-old C57Bl/6.lpr autoimmune mice were used as positive control. Briefly, 96-well plate were coated overnight with either spike protein (0.2µg/mL) or calf thymus DNA (0.1mg/mL; after precoating with poly-L-lysin 0.05mg/mL). After blocking with PBS-BSA, the diluted serum samples were incubated on the plate for 2 hours at room temperature. Plate-bound IgG were revealed using an alkaline phosphatase-conjugated anti-mouse IgG (1:5000). For anti-dsDNA IgG measurement, the serum of an autoimmune mice was used as a reference standard and the result expressed in unit per mL.ResultsAfter immunization, the phenotype of control and spike-immunized mice was clinically similar. All the immunized mice were exempt of proteinuria (Figure 1B). Only the mice which were injected with spike protein developed measurable amounts of anti-spike IgG as determined by ELISA, while those injected with PBS and the C57Bl/6.lprmice did not (Figure 1C). None of the mice in the immunization group developed measurable anti-dsDNA IgG (Figure 1D).ConclusionIn our study, the repetitive exposure of C57Bl/6 mice to the SARS-CoV-2 spike antigen did not lead to autoimmunity. Although these results are reassuring, large scale epidemiological studies are needed to evaluate the incidence of autoimmune diseases in individuals with multiple exposure to SARS-CoV-2 antigens.Reference[1]K. Tsumiyama, Y. Miyazaki, S. Shiozawa, Self-Organized Criticality Theory of Autoimmunity, PLOS ONE. 4 (2009) e8382.Figure 1.Acknowledgements:NIL.Disclosure of InterestsMarc SCHERLINGER Speakers bureau: Amgen, Sandoz, Nordic Pharma, Jean Sibilia Speakers bureau: Roche, Chugai, Bristol-Myers Squibb, UCB, GSK, LFB, Actelion, Pfizer, MSD, Novartis, Amgen, Abbvie, Sandoz, Gilead, Lilly, Sanofi Genzyme, Janssen, Mylan, Galapagos, Sobi., Grant/research support from: Pfizer, BMS, Roche, MSD, George Tsokos: None declared, Jacques-Eric Gottenberg Speakers bureau: AbbVie, BMS, CSL Behring, Galapagos, Gilead, Lilly, Roche-Chugai, Pfizer, Sanofi, and UCB., Grant/research support from: BMS.

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Abstract Background The Roche Cobas chemistry analyzer’s hemolysis index (HI) check function can directly report hemoglobin (Hb) concentrations. We aimed to validate the HI check function for the measurement of plasma cell-free Hb. Methods Plasma samples (6 μl) were taken by the analyzer and diluted in normal saline to measure the absorbance for Hb at 570 and 600 nm. Hb concentrations were calculated based on the molar extinction coefficient. Imprecision, lower limit of quantification (LLOQ), and analytical measurement range (AMR) of the assay were evaluated. The accuracy was determined by comparing the results between the new method and an existing spectrophotometric method. We further studied interference of icterus and lipemia and carryover. The performance of the assay in proficiency testing was also evaluated. The reference range was transferred from the existing method. Results Within-run and total CVs were 1.7%–4.2% and 2.1%–7.0%, respectively (n = 20). The LLOQ was 11 mg/dL (CV = 8.1%) with the upper limit of AMR of 506 mg/dL. The results of the new method correlated well with the existing reference assay: Y (new method) = 0.974 x (reference method) + 4.9, r = 0.9990, n = 52. Bilirubin with a concentration up to 60 mg/dL and lipemic index up to 389 did not show significant interference. No significant carryover was detected. The average standard deviation index in proficiency testing was 0.03 ± 0.29. The reference range was &lt;22 mg/dL. Conclusions Plasma cell-free Hb measurement using the HI check function meets the analytical requirements of the plasma cell-free Hb assays. It is simple and cost-effective.

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Background: Thermal waters have been showing different beneficial effects on the skin due to their physicochemical composition. The beneficial effect of thermal water in the treatment of some skin diseases may thus justify its use as an active ingredient in cosmetic formulations. The main objective of this work was to demonstrate the potential of incorporating thermal water as an active ingredient in cosmetic formulations. (2) Methods: A descriptive literature review was carried out by the analysis of scientific articles in PubMed and Google Scholar databases. Twelve thermal spring waters were found (Avène, Blue Lagoon, Comano, Cró, Dead Sea, La Roche-Posay, Monfortinho, Saint-Gervais, Salies-de-Béarn, São Pedro do Sul, Uriage and Vichy) with potential as an active in cosmetic products, demonstrated through in vitro studies evaluating the different activities/properties and clinical trials in healthy volunteers or with skin pathologies. (3) Results: For these studies, in natura thermal water as well as incorporated in cosmetic formulations were used. In in vitro studies, most thermal waters have been shown to have activities on membrane fluidity, skin barrier repair, antiradical, antioxidant, anti-inflammatory and immunomodulatory properties, proliferative activity, regulation of processes involved in ageing and moisturizing properties. In clinical trials, cosmetic thermal waters reduced skin discomfort through their soothing and exhibited moisturizing and anti-irritant properties. (4) Conclusions: The effect of thermal waters on the skin and the absence of side effects reported in different studies allows them to be used as an adjuvant or in the treatment of various skin disorders and may play an important role in the cosmetics industry. However, further clinical trials are needed to assess their effectiveness and safety.

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This work describes the treatment with total parenteral nutrition (TPN) of a 37-day cloned calf after suffering ruminal acidosis by ingestion of milk. Cells for SCNT were obtained by using a bicistronic vector for human lysozyme and lactoferrin. We obtained 7 embryos and 2 pregnancies. Only one fetus was born alive weighing 47 kg and it was presented to the neonatal unit of INTA showing a deep depression, diarrhoea, dehydration (10%), hyperthermia and inability to stand (Table 1). A 20-cm-length 20-Ga-diameter catheter (Arrow) was placed in the jugular vein. Blood samples from catheter and brachial artery showed leukocytosis, hypoglycemia and metabolic acidosis (Table 1). Esophageal tube was placed to remove 5 L of ruminal content and for the administration of 2 L of a solution of sodium bicarbonate (40 g L–1 of water). Saline (NaCl, 9 g L–1), sodium bicarbonate (8.4 g/100 mL) and 10% dextrose were administered IV until dehydration; blood pH and glucose were corrected. Ceftriaxone 1 g IM/12 h (Acantex, Roche) to prevent bacterial translocation, 1.175 mg of flunixin meglumine (PharmaVet) as anti-endotoxic dose and 80 mg of ranitidine IV/12h (Vetanco) to prevent laminitis were also administered. Two litres of bovine plasma were administered during the first 2 days and, after this, we began with a TPN regimen due to lack of sucking reflex and animal anorexia. Kabiven (Fresenius Kabi AV) was administered at 1 g of lipids/kg/24 h by a regimen of 18 drops min–1 to prevent hyperlipidemia at the recommended dose for humans. For this reason, we also administered dextrose 25% 12.5 drops min–1 and amino acids 11.5% 504 mL (Rivero), to reach a dose of 10 g/kg/24 h and 2 g/kg/24 h, respectively. Saline (NaCl, 9 g L–1) and vitamin complex (Rivial Paediatrics, Rivero) was also administered to cover water and vitamin requirements. The TPN therapy lasted 24 days during which the animal regularized its metabolic functions, reversed signs of ruminal acidosis and learned to eat a balanced ration and hay. To our knowledge, no information is available on such a long period of TPN in bovine neonates. This work shows that TPN can increase the survival chances of high risk animals and thus the final efficiency of cloning and transgenesis. Table 1.Clinical and haematological responses to 24-day total parenteral nutrition in a 37-day cloned female calf The authors thank Fresenius Kavi and Rivero Laboratories for their support.

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L’exploitation intensive des ressources en eaux souterraines du bassin Chougafiya a largement influencé le fonctionnement hydrodynamique de la nappe phréatique. Elle a provoqué une inversion du gradient hydraulique et une invasion des eaux minéralisées en provenance de la nappe du Sabkhas El Batten. Afin de mettre en évidence l’effet de cette intrusion salée sur l’hydrodynamisme de la nappe phréatique, une étude multidisciplinaire basée sur les méthodes de l’hydrogéologie et la géochimie a été menée. L’étude hydrochimique a montré que le faciès des eaux évolue entre un pôle chloruré sulfaté calcique et chloruré sodique. Les principaux phénomènes géochimiques intervenant dans l’acquisition de la charge saline sont liés à l'interaction eau-roche (dissolution des minéraux évaporitiques) et à l'échange cationique. Cependant, la composition chimique des eaux minéralisées situées dans les parties centrale et méridionale du bassin d'étude est nettement influencée par un processus de mélange avec l'eau de la nappe du Sabkhas. Le traçage naturel des eaux au moyen des isotopes stables (18O, 2H) a permis d’identifier des eaux d’origine météorique et à caractère non évaporé, tandis que les eaux à caractère évaporé s’alignent selon une droite de mélange avec l’eau souterraine de la Sabkhas. L’évolution des teneurs en 3H et 14C témoigne d’une forte contribution des eaux récentes à la recharge de la nappe phréatique dans les secteurs nord et ouest, alors que les faibles activités enregistrées au centre et à l’est de la plaine reflètent la présence d’une lithologie peu perméable. L’étude des isotopes du carbone a permis de comprendre le schéma du fonctionnement de la nappe étudiée et d’estimer le temps de séjour des eaux. D’ailleurs, les âges calculés varient de l’actuel jusqu’à 7 814 ans BP. L’enrichissem*nt isotopique en carbone-13 qui apparaît au fil de l’écoulement est révélateur de phénomènes d’échanges isotopiques avec la matrice carbonatée de l’aquifère.

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Abstract Background Body fluid samples (BFs) with low pH have demonstrated underrecovery of enzyme activity. The aims of this study were (1) measure the frequency of abnormal pH in BFs received for clinical chemistry testing, (2) calculate recovery of analytes in BFs undergoing pH titration, and (3) investigate the mechanism of pH interference. Methods Residual BFs submitted for physician-ordered testing to the Central Clinical Laboratory (Mayo Clinic) were utilized for this study. BFs were centrifuged 10 minutes (3,200 rpm) prior to pH measurement (pH meter, Accumet X15; Fisher Scientific) and analysis. A Roche Cobas c501 (Roche Diagnostics) analyzer was used to measure amylase, lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine, glucose, albumin, total protein, bilirubin (all Roche Diagnostics reagents), and lipase (Sekisui Diagnostics). pH was measured in 122 BFs received between 10/23/2018 and 10/26/2018 within 24 hours of receipt. pH titration was performed using pooled BFs of similar source/type. Each pool was titrated over a range of pH (≤2 to ≥10) in 1-unit increments by addition of a fixed volume of acid (6M HCL) or base (6M NaOH). Analytes were measured before and after acid/base addition. Average percent recovery was calculated (measured/expected × 100%) from n = 3 to 9 pH measurements. A low pH BF pool was spiked (<5% by volume) with a high-concentration BF to investigate mechanism of enzyme underrecovery. Additionally, low pH BF pool was neutralized by addition of base and % recovery calculated. For all titration/spiking experiments, a control sample having the same volume of diluent (7% bovine serum albumin or saline) was used to account for the effects of dilution. Results BFs received (n = 122) had mean (SD) pH = 8.0 (0.6) with 6.6% (n = 8) having pH <7.0 and 2.5% (n = 3) having pH <6.0. All recovery (%) and pH data are expressed as mean (range) values. Amylase recovery (initial pH = 8.4-8.9) was 1.3 (0.9-2.2)% at pH = 1.6 (1.0-2.2) (n = 6), 55.6 (37.4-67.0)% at pH = 4.4 (3.5-5.4) (n = 6), 81.4 (74.0-85.2)% at pH = 6.2 (6.1-6.5) (n = 3), 90.1 (84.4-95.4)% at pH = 7.5 (7.3-7.7) (n = 3), 98.8 (96.2-101.7)% at pH = 10.0 (9.8-10.2) (n = 3), and 14.5 (1.4-38.9)% at pH = 11.9 (11.5-12.1) (n = 3). LDH recovery (initial pH = 8.2-8.5) was 1.4 (0.1-3.7)% at pH = 1.7 (1.0-2.4) (n = 6), 18.3 (0.5-39.7)% at pH = 4.5 (3.5-5.4) (n = 6), 63.6 (54.9-68.4)% at pH = 6.2 (6.1-6.5) (n = 3), 85.9 (80.2-90.7)% at pH = 7.2 (6.8-7.4) (n = 3), 46.1 (36.4-61.0)% at pH = 10.0 (9.8-10.2) (n = 3), and 9.8 (0.0-28.9)% at pH = 11.3 (10.4-12.1) (n = 3). Lipase recovery (initial pH = 8.2-8.9) was 10.4 (<1-18)% at pH = 1.8 (1.0-2.4) (n = 6), 73.9 (62.6-85.5)% at pH = 4.2 (3.4-5.2) (n = 3), 92.4 (90.8-93.5)% at pH = 6.2 (6.0-6.5) (n = 3), 96.3 (95.6-96.8)% at pH = 7.3 (6.8-7.7) (n = 3), and 92.9 (89.3-96.8)% at pH = 9.9 (9.8-10.1) (n = 3) and 80.9 (0.0-78.6)% at pH = 11.3 (10.4-12.1) (n = 3). Creatinine, BUN, albumin, glucose, total protein, and bilirubin recovery were 97.9 (92.2-102.9)%, 100 (96.6-103.1)%, 99.9 (97.6-102.4)%, 100.2 (97.8-103)%, 100.2 (98.7-101.9)%, and 100.1 (92.-105.9)%, respectively, between pH 1.3-11.7 (n = 9). Recovery of amylase, LDH, or lipase was <1% after spiking enzymes into BF pool with pH <3. pH adjustment to normal (pH = 8.6-9.0) also resulted in recovery of <1%. Conclusion Enzyme activity in BFs was not affected (90%-110% recovery) when pH = 7.4-8.5 for LDH, pH = 7.3-10.2 for amylase, pH = 6.0-9.9 for lipase, and pH = 1.3-11.7 for all other analytes. An irreversible loss of enzyme activity occurs at low pH. Few clinical BFs have pH <7.0, but laboratories should have awareness.

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Background:Golimumab is a human monoclonal antibody directed against TNFα in its soluble and transmembrane forms. It can be used subcutaneously or intravenously and has shown efficacy for use in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS).Objectives:The aim of this study was to evaluate the efficacy, safety, and cumulative survival of golimumab in patients with RA, PsA and AS from different rheumatology centers in Argentina.Methods:We performed a longitudinal study of consecutive adults with RA (ACR/EULAR 2010 criteria), PsA (CASPAR criteria) and AS (ASAS 2009 criteria), who have started treatment with subcutaneous or intravenous golimumab according to medical indication in each center. Data was obtained by review of medical records. Sociodemographic and clinical data, musculoskeletal manifestations, comorbidities, previous treatments were recorded. In reference to golimumab treatment, start date, route of administration and concomitant treatments were identified. Disease activity was assessed using DAS28 for RA patients, DAPSA and MDA for PsA and BASDAI for AS. The presence of adverse events (AE) was recorded. If golimumab was stopped, date and cause was documented. Patients were followed up until golimumab discontinuation, loss of follow-up, or study completion (November 30, 2020). Statistical analysis: Chi2 test or Fischer exact test and T test or Mann Whitney and ANOVA or Kruskal Wallis, as appropriate. The incidence of EA was assessed in events every 100 patient/year. Kaplan-Meier curves and log Rank analysis. Cox proportional regression.Results:One hundred eighty two patients were included, 116 with a diagnosis of RA, 30 with PsA and 36 with AS. Most of them (70.9%) were female with a median (m) age of 55 years (IQR 43.8-64) and m disease duration of 7 years (IQR 4-12.7) at treatment initiation. Al least one prior biological DMARD or a small molecule was received by 63 patients (34.6%). The most frequent indication cause was conventional DMARD failure. In 94.8% of the patients Golimumab was administered subcutaneously, and in 80.8% in association with conventional DMARDs, the most frequently used was methotrexate. Total follow-up was 318.1 patients/year.Golimumab treatment showed clinical improvement in all three groups of patients. In RA patients DAS28 significantly decreased during the first 12 months of follow-up, m 5.9 (IQR 4.9-6.6) at baseline, 3.8 (IQR 2.6-4.6) at 6 months and 2.8 (IQR 2.1-3.6) at 12 months, p <0.0001. In PsA, m DAPSA-ESR value was 32.2 (IQR 24.2-47.7), 10.1 (IQR 5.8-18.3) and 11.2 (IQR 3.4-24) at baseline, 6 and 12 months, respectably (p <0.0001). In AS, m BASDAI was 6.2 (IQR 4.8-7.3), 2.8 (IQR 1.7-4.1) and 2.2 (IQR 1.1-3.2), at baseline, 6 and 12 months respectively (p <0.0001).The incidence of adverse events was 6.6 per 100 patients/year, being infections the most frequents ones. During follow-up, 50 patients (27.5%) discontinued golimumab, the most frequent cause was treatment failure (68%), followed by lack of health insurance (16%) and adverse events (10%). Golimumab persistence was 79% and 57.6% at 12 and 24 months, respectively. Treatment survival was 50.2 months (95% CI 44.4-55.9). Patients who had received prior treatment with biological DMARDs or small molecules showed lower survival (Figure 1). In the multivariate analysis, adjusting for age, sex and disease duration, those patients showed twice the risk of suspending treatment (HR 2.01, 95% CI 1.1-3.7).Figure 1.Golimumab survival according to prior b-DMARD o small molecule treatment.Conclusion:Golimumab treatment in real life patients in Argentina has shown good efficacy and safety. Drug survival was over 4 years and almost 80% were still using golimumab after one year. Prior treatment with other b-DMARDs o small molecules was associated with lower treatment survival.Disclosure of Interests:Carolina Ayelen Isnardi Speakers bureau: Bristol Myers Squibb, Janssen, Grant/research support from: Pfizer, Emma Estela Civit De Garignani Speakers bureau: Abbvie, Novartis, Agustín García Ciccarelli Speakers bureau: Janssen, Novartis, Consultant of: Novartis, Grant/research support from: Janssen, Novartis, Jimena Sanchez Alcover: None declared, Rodrigo Garcia Salinas Speakers bureau: Abbvie, AMGEN, Bristol-Myers Squibb, Eli Lilly, GSK, Janssen Cilag, Montpellier-UCB, Novartis, Roche – Genentech, Sanofi, Merck Serono., Sebastian Magri Speakers bureau: Abbvie, AMGEN, Bristol-Myers Squibb, Eli Lilly, GSK, Janssen Cilag, Montpellier-UCB, Novartis, Roche – Genentech, Sanofi, Merck Serono., Eduardo Albiero Consultant of: Janssen, Carla Gobbi Speakers bureau: Pfizer, Consultant of: Pfizer, Janssen, Edson Velozo Speakers bureau: Janssen, Novartis, Pfizer, Consultant of: Abbvie, Janssen, Novartis, Grant/research support from: Janssen, Novartis, Pfizer, Enrique Soriano Speakers bureau: AbbVie, Novartis, Bristol MS, Novartis, Eli Lilly, Genzyme, Pfizer, Amgen, and Roche, Consultant of: Novartis, AbbVie, Pfizer, Eli Lilly, Sanofi, Sandoz, Amgen., Grant/research support from: Roche, Novartis, AbbVie, Glaxo Smith Kline, BMS, Martín Brom: None declared, Johana Zacariaz Grant/research support from: Bristol Myers Squibb, Ingrid Strusberg Speakers bureau: Gema Biotech SAU, BMS, Abbvie, Consultant of: Gema Biotech SAU, Abbvie, Janssen, Grant/research support from: Abbvie, Lilly, Galápagos, Servier, GSK, Merck Serono, Marcos BARAVALLE Speakers bureau: Montepellier, Consultant of: Abbvie, Janssen, Grant/research support from: Abbvie, Lilly, Galápagos, Servier, GSK, Merck Serono, Sol Castaños Speakers bureau: Abbvie, Lilly, Galápagos, Servier, GSK, Merck Serono, Liliana Morales Speakers bureau: Lilly, Consultant of: Janssen, Grant/research support from: Abbvie, Lilly, Galápagos, Servier, GSK, Merck Serono, Sergio Paira: None declared, Romina Calvo: None declared, Alberto Ortiz: None declared, Rodolfo Perez Alamino Speakers bureau: Pfizer, Abbvie, Amgen, Bristol-Myers-Squibb, Lilly, Janssen, Novartis, Hernan Maldonado Ficco Speakers bureau: Pfizer, Abbvie, Jansen, Novartis, Bago, Bristol, Eli Lilly., Consultant of: Pfizer, Abbvie, Novartis, Jansen, Bago, Eli Lilly., Gustavo Citera Speakers bureau: Abbvie, BMS, Lilly, Jansen, Gema, Pfizer, Roche, Grant/research support from: Pfizer

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Abstract Introduction/Objective Histologic frozen section analysis is typically used for parathyroid tissue identification during parathyroidectomy when needed, in conjunction with a rapid intraoperative plasma parathyroid hormone (PTH) assay to confirm falling levels of circulating PTH after parathyroid excision. As an alternative to frozen section consults, we hypothesize that automated analysis of intraoperative fine needle aspiration (ioFNA) tissue samples using a rapid PTH immunoassay can accurately identify parathyroid tissue and reduce the need for frozen section consults. Methods A rapid PTH immunoassay (Elecsys PTH STAT; Roche Diagnostics, Indianapolis, IN), currently used for intraoperative plasma samples, was validated for FNA samples on a Cobas® e411 using tissue aspirates of ex vivo parathyroid and control specimens rinsed in 1mL saline. ioFNA PTH results during parathyroidectomy were then prospectively assessed for accuracy over a 4-month period by comparing values to final histopathologic diagnoses. The number of frozen section consults requested was compared to a 5-month period prior to the availability of the ioFNA PTH assay. Results Ninety patients underwent parathyroidectomy (128 excised parathyroids) during the study period, performed by a single experienced endocrine surgeon. Indications included primary (81/90), tertiary (5/90), and recurrent (4/90) hyperparathyroidism. Thirty-nine cases (55.5 excised parathyroids) were performed after the availability of the ioFNA PTH assay. ioFNA samples were sent for PTH analysis in 7/39 cases (18%; 12 samples total) and had a sensitivity/specificity of 100% (parathyroid [n=7] PTH values 1968 - &gt;5000pg/mL; non-parathyroid [n=5] PTH values &lt;2 - 16pg/mL). Parathyroidectomies requiring frozen section consult significantly decreased from 41% (21/51 cases; 40 specimens) to 10% (4/39 cases; 9 specimens) with the availability of the ioFNA PTH assay (p&lt; 0.05, Fisher exact test). Conclusion Analysis of ioFNA tissue samples using an automated rapid PTH immunoassay can accurately identify parathyroid tissue and can be used as an alternative to frozen section consult when needed.

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Background:In recent years, diverse compounds for intra-articular administration were brought into the market with a subsequent significant and heterogeneous literature production. Understanding the efficacy of intra-articular therapies (IAT) on pain implies bearing in mind the related placebo (PBO) effect. To date, most studies analyzing it were focused on the compound being administered rather than the route of administration.Objectives:We aimed at evaluating the size of the PBO effect after intra-articular injections.Methods:We conducted an overview of systematic reviews (SRs) including randomized-controlled trials (RCTs) of frequently used IAT. SRs with a saline solution PBO arm and high-confidence results according to the AMSTAR-2 tool were selected for analysis.Data on the change in pain in the PBO arms from baseline to 3-6 and 12-16 weeks after the IA procedure was extracted. The standardized mean differences (SMD) from baseline were calculated as the ratio between the size of the intervention effect in each study and the variability observed in that study. A meta-analysis was then performed using an inverse-variance random-effects model in Review Manager 5.3. The overall effect sizes obtained refer to versions of the SMD, which corresponds to the Hedges’ (adjusted) g. e.g. a “g” of 1 indicates the two groups being compared differ by 1 standard deviation and so on.Results:Two SR were included comprising 50 RCTs, 44 not meeting inclusion criteria were excluded so pain, measured by visual analogue scale (VAS) and Lequesne index, was retrieved from 6 RCT.At 3-6 weeks, an SMD [95%CI] = 0.74 [0.47-1.00] was found. One study showing too large an effect was excluded after conducting sensitivity analysis resulting in a significant reduction of heterogeneity with an SMD = 0.62 [0.45-0.79] (Fig.1). At 12-16 weeks, we found a SMD = 0.33 [0.13-0.52] (Fig.2)Figure 1.Forest plot for intra-articular PBO effect at 3-6 weeks after injection.Figure 2.Forest plot for intra-articular PBO effect at 12-16 weeks after injection.Using the interpretation suggested by Cohen1, our results would confirm a moderate to large effect of IA saline (PBO) at 3-6 weeks with a subsequent reduction to a small but persistent effect at 12-16 weeks.Conclusion:Our results showed a moderate to large short-term effect of intra-articular PBO that persisted on the mid-term although reduced. Based on these findings we suggest this effect should be considered when assessing the efficacy of IAT in RCTs and also in clinical practice where it could be maximized as well.References:[1]Cohen J. Statistical Power Analysis in the Behavioural Sciences (2nd edition). Hillsdale (NJ): Lawrence Erlbaum Associates, Inc., 1988.Disclosure of Interests: :Sebastian C Rodriguez-García Speakers bureau: Novartis Farmaceutica, S.A., Merck Sharp & Dohme España, S.A., Sanofi Aventis, UCB Pharma, Raul Castellanos-Moreira Speakers bureau: Lilly, MSD, Sanofi, UCB, Jacqueline Uson Jaeger: None declared, Esperanza Naredo: None declared, Loreto Carmona Grant/research support from: Novartis Farmaceutica, SA, Pfizer, S.L.U., Merck Sharp & Dohme España, S.A., Roche Farma, S.A, Sanofi Aventis, AbbVie Spain, S.L.U., and Laboratorios Gebro Pharma, SA (All trhough institution)

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Maternal oocyte Cyclin B1 mRNA is known to be stored in the cytoplasm with a short poly(A) tail and be translationally dormant at GV stage. During maturation, Cyclin B1 poly(A) tail is elongated by a process called cytoplasmic polyadenylation and driven by A/U-rich cis-acting elements in its 3′ untranslated region (UTR) known as cytoplasmic polyadenylation elements (CPEs). The objective of this study was to elucidate whether GV-stage bovine oocytes possess a stockpile of Cyclin B1 mRNA stored with a short a poly(A) tail that is elongated during maturation by CPE regulation. The mRNA poly(A) tail length was measured by Rapid Amplification of cDNA Ends Polyadenylation test (Race-PAT) on oocytes (n=100) at the GV stage and 3, 5, 8, 10, 15, 20, and 25h of in vitro maturation. The mRNA poly(A) tail length was also measured in triplicate (n=20) on cold oocytes in GV (all manipulations on ice), warm oocytes in GV (ovaries transported in warm saline and manipulations on ice) and warm+2h 30min oocytes in GV (oocytes left for an additional 2h and 30min at room temperature). To assess for variation in mRNA quantity, Cyclin B1 mRNA level was quantified by real-time PCR (Lightcycler, Roche, Indianapolis, IN, USA) in cold, warm or warm+2h 30min GV oocytes (n=20). The data were treated as factorial design, using treatment and type of RT as factors, and analysed by ANOVA (SAS Inst., Cary, NC, USA). Differences between means were checked using Tukey’s test. Oocyte Cyclin B1 transcript show two different 3′ UTRs. These transcripts had the same ORF but different 3′ UTR lengths because of an alternative nuclear polyadenylation element AAUAAA (NPE). The longest form (Cyclin B1L) that possessed a putative CPE (UUUUAAUAAA) fused to the last NPE was studied. In warm GV oocytes, Cyclin B1L had a long poly(A) tail of 100 adenosine residues, and this length did not change during in vitro maturation. Interestingly, we found that Cyclin B1L showed an expected short poly(A) tail when the ovaries and the oocytes were transported and manipulated on ice. We showed that Cyclin B1L mRNA is cytoplasmically polyadenylated (addition of 75 adenosine residues) between the time of collection and the end of manipulation. This lengthening is most probably sufficient to promote translation. There was no significant difference between the Cyclin B1 mRNA quantity of cold oocytes or warm oocytes when the oligo used for the reverse transcription was either dt or decamers. Therefore, we believe that the increase in poly(A) tail length is not the result of Cyclin B1L mRNA degradation in cold oocytes or de novo transcription in warm oocytes. We report for the first time that Cyclin B1L cytoplasmic polyadenylation is carried out well before the beginning of in vitro maturation in bovine oocytes when ovaries are transported from the slaughterhouse in warm saline. Studying the real early mechanisms leading to resumption of meiosis in bovine oocytes is complicated by Cyclin B1 polyadenylation occurring prior to in vitro maturation. (Supported by NSERC.)

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Background:Glucocorticoids (GCs) are a key treatment for the autoimmune rheumatic diseases; however, they produce numerous physical and psychological side effects.1 The Outcome Measures in Rheumatology (OMERACT) Glucocorticoid Working Group have identified that there are no Patient Reported Outcome Measures (PROMs) for assessing the impact of systemic GC therapy across multiple rheumatic diseases from the patient’s perspective.2,3Objectives:The aim is to explore the impact of GCs on the symptoms and health-related quality of life of adults with rheumatic inflammatory diseases, to inform items for inclusion in a PROM. Key considerations will include patient perceptions of GC therapy at diagnosis and over the course of treatment, for use in future randomised controlled trials or in clinical practice.Methods:An international steering committee comprising researchers, rheumatology clinicians, methodologists and patient partners in the UK, Australia and USA developed an initial conceptual framework informed by a review of the literature. Semi-structured interviews were conducted in each country with patients who had an autoimmune rheumatic disease and had received GC therapy. The interviews explored salient aspects of health-related quality of life associated with being treated with GCs.Results:Interviews have been completed in three continents with patients who had a range of demographic features, rheumatological conditions and duration and dosage of GC therapy. Figure 1 shows the initial conceptual framework for developing the GC PROM (Steroid PRO).Figure 1.Conclusion:This conceptual framework will act as an evolving guide in the development of a PROM for assessing patients’ perspectives of systemic glucocorticoid therapy. Future work will include inductive analysis of qualitative transcripts to inform candidate questionnaire items, cognitive interviewing, linguistic translatability assessment, and an international validation survey to define the final PROM questionnaire and its measurement properties.References:[1]Cheah JTL, Robson JC, Black RJ, et al. The patient’s perspective of the adverse effects of glucocorticoid use: A systematic review of quantitative and qualitative studies. From an OMERACT working group. Semin Arthritis Rheum. 2020 Oct; 50(5):996-1005.[2]Black RJ, Robson JC, Goodman SM, et al. A Patient-reported Outcome Measure for Effect of Glucocorticoid Therapy in Adults with Inflammatory Diseases Is Needed: Report from the OMERACT 2016 Special Interest Group. J Rheumatol. 2017; 44(11):1754-8.[3]Cheah JTL, Black RJ, Robson JC, et al. Toward a Core Domain Set for Glucocorticoid Impact in Inflammatory Rheumatic Diseases: The OMERACT 2018 Glucocorticoid Impact Working Group. J Rheumatol. 2019; 46(9):1179-1182.Disclosure of Interests:Susan Bridgewater Grant/research support from: Grant from Vifor Pharma for an independent investigator-led study to develop a PRO for steroids, Jill Dawson: None declared, Mwidimi Ndosi: None declared, Rachel J Black: None declared, Jonathan T.L. Cheah: None declared, Emma Dures: None declared, Nilasha Ghosh: None declared, Elizabeth A Hoon: None declared, Iris Navarro-Millan Consultant of: Received consultant fees from SOBI, Diyu Pearce-Fisher: None declared, Pamela Richards: None declared, Carlee Ruediger: None declared, Christine Silverthorne: None declared, Joanna Tieu Grant/research support from: Vifor Pharma, Sarah Mackie Consultant of: Consultancy on behalf of institution for Roche/Chugai, Sanofi, AbbVie and AstraZeneca, Grant/research support from: Educational grant from Roche to attend EULAR2019, Susan Goodman: None declared, Catherine Hill: None declared, Joanna Robson Speakers bureau: Vifor Pharma for educational webinar, Grant/research support from: Grant from Vifor Pharma for an independent investigator-led study to develop a PRO for steroids

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Abstract Introduction: Prognosis remains poor for patients (pts) with R/R cHL who relapse after or are ineligible for autologous stem cell transplant (ASCT). Programmed death 1 (PD-1) inhibitors are an effective therapeutic option for such pts. Previous analyses from the phase 2 KEYNOTE-087 (NCT02453594) trial demonstrated that monotherapy with the PD-1 inhibitor pembrolizumab (pembro) had effective antitumor activity and acceptable safety in pts with R/R cHL, leading to FDA approval for adult and pediatric pts with R/R cHL who relapsed after ≥3 prior lines of therapy. However, durability of response after pembro, its relationship with response depth, and safety of treatment discontinuation for pts achieving complete response (CR) remain salient clinical questions. We present efficacy and safety from KEYNOTE-087 after ~5 y of follow-up. Methods: Pts with R/R cHL that progressed after ASCT and subsequent brentuximab vedotin (BV, cohort 1), progressive disease (PD) after salvage chemotherapy and BV therapy without ASCT (cohort 2), or PD after ASCT without subsequent BV therapy (cohort 3) received pembro 200 mg every 3 wks for up to 2 y. Pts who discontinued treatment after initial CR and relapsed were eligible to receive up to 17 additional cycles (~1 y) of pembro (2nd course). Primary end points: ORR per blinded independent central review (BICR), and safety. Additional end points: ORR per Lugano classification, DOR, and PFS by BICR; OS and 2nd-course ORR per investigator. DOR, PFS, and OS were estimated by Kaplan-Meier (K/M) method. Data cutoff was March 15, 2021. Results: At data cutoff, median time from first dose to date of death or data cutoff was 62.9 mo (range, 1.0-68.7). In the total population (N=210), 46 pts completed 2 y of treatment and 164 pts discontinued (primarily due to progressive disease [n=86]). ORR was 71.0% (95% CI, 64.8-77.4; CR, 27.6%; partial response [PR, 43.8%]); results were similar per Lugano classification (ORR, 73.3% [95% CI, 66.8-79.2]; CR, 32.9%; PR, 40.5%). ORR was 84.1% (CR, 36.2%; PR, 47.8%) in cohort 1 (n=69), 67.9% (CR, 28.4%; PR, 39.5%) in cohort 2 (n=81), and 68.3% (CR, 35.0%; PR, 33.3%) in cohort 3 (n=60). In the total population, median DOR was 16.6 mo (95% CI, 11.8-27.1). Four pts (24.8% per K/M method; cohort 1, n=1; cohort 2, n=0; cohort 3, n=3) had response ≥60 mo. In the total population, median PFS was 13.7 mo (95% CI, 11.1-19.4) and the 5-y PFS rate was 14.2%: 16.4 mo (95% CI, 11.3-27.6) in cohort 1, 11.1 mo (95% CI, 7.5-13.7) in cohort 2, and 19.7 mo (95% CI, 10.8-27.3) in cohort 3. In the total population, median OS was not reached (NR) and the 5-y OS rate was 70.7%: 71.3% in cohort 1, 69.2% in cohort 2, and 71.5% in cohort 3. Of 58 pts in the total population who achieved CR (Table), median DOR was NR (95% CI, 16.1-NR) and 4 pts (51.6% per K/M method; cohort 1, n=1; cohort 2, n=0; cohort 3, n=3) had a response ≥60 mo. Median PFS was 56.5 mo (95% CI, 21.7-NR) and 5-y PFS rate was 44.3%; median OS was NR and 5-y OS rate was 82.8%. Ten pts with CR received allogeneic stem cell transplant (allo-SCT) and 48 pts with CR did not undergo allo-SCT. Of 20 pts who received 2nd-course treatment (cohort 1, n=10; cohort 2, n=7; cohort 3, n=3), 10 pts (cohort 1, n=2; cohort 2, n=6; cohort 3, n=2) completed 17 additional cycles of treatment; 1 pt each from cohort 1 and cohort 3 were mid-treatment. ORR based on 19 evaluable pts from cohorts 1-3 was 73.7% (95% CI, 48.8-90.9). Median DOR for 2nd course was 15.2 mo (95% CI, 3.9-32.9), and 1 pt (13.7%) had a second response ≥36 mo. Treatment-related adverse events (AEs) of any grade occurred in 72.9% of pts; most common (&gt;10%) were hypothyroidism (14.3%), pyrexia (11.4%), and fatigue (11.0%). Grade 3-4 treatment-related AEs occurred in 12.9% of pts; neutropenia (n=5), diarrhea and pericarditis (n=2, each) occurred in ≥2 pts. All-cause grade 5 AEs (n=3) were due to acute graft-versus-host disease, infection, and septic shock; these AEs were not considered treatment related. Conclusions: With median of follow-up &gt;5 y, pembro monotherapy demonstrated sustained antitumor activity in pts with R/R cHL. ORRs were high and responses durable in the overall population and in those with varied treatment histories. Pts with CR had especially durable responses, and in those relapsing from CR, 2nd-course pembro frequently re-induced sustained response. Safety was manageable with no new signals. Figure 1 Figure 1. Disclosures Armand: Merck & Co., Inc.: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; Affimed: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Infinity: Consultancy; ADC Therapeutics: Consultancy; Celgene: Consultancy; Morphosys: Consultancy; Daiichi Sankyo: Consultancy; Miltenyi: Consultancy; Tessa: Consultancy; GenMab: Consultancy; C4: Consultancy; Enterome: Consultancy; Regeneron: Consultancy; Epizyme: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Roche: Research Funding; Tensha: Research Funding; Otsuka: Research Funding; Sigma Tau: Research Funding; IGM: Research Funding; Kite: Research Funding. Zinzani: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kyowa Kirin: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Portola: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ImmuneDesign: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck Sharp & Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lee: Oncternal: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Seagen: Research Funding; Century Therapeutics: Consultancy; Guidepoint: Honoraria; Aptitude Health: Honoraria; BMS: Honoraria, Research Funding; Janssen: Honoraria; Pharmacyclics: Research Funding. Johnson: AbbVie: Consultancy, Research Funding; Merck: Consultancy; Roche: Consultancy, Honoraria; BMS: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Brice: MSD: Research Funding; Amgen: Other: Travel/accommodations/expenses; Takeda: Research Funding; Roche: Other: Travel/accommodations/expenses. Radford: Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Current holder of individual stocks in a privately-held company; ADC Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Honoraria, Speakers Bureau; BMS: Honoraria. Ribrag: Roche: Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Argen-X: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astex Pharmaceuticals: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; PharmaMar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Epizyme: Honoraria, Research Funding; MSD Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Infinity Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Molin: Roche: Honoraria; MSD: Honoraria; BMS: Honoraria; Takeda: Honoraria. Vassilakopoulos: AbbVie: Consultancy, Honoraria; GlaxoSmithKline: Honoraria, Other: Travel; Amgen: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Merck: Honoraria, Research Funding; Genesis Pharma: Consultancy, Honoraria, Other: Travel; Roche: Consultancy, Honoraria, Other: Travel; Takeda: Consultancy, Honoraria, Other: Travel, Research Funding; Integris: Honoraria; AstraZeneca: Honoraria; Pfizer: Research Funding; Dr. Reddy's: Research Funding; Karyopharm: Research Funding. Tomita: Kyowa Kirin: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria, Research Funding; Astellas Pharma: Honoraria, Research Funding; Nippon Shinyaku: Consultancy, Honoraria, Research Funding; Janssen Pharmaceutical: Honoraria; Zenyaku Kogyo: Honoraria; AbbVie GK: Honoraria; Bristol Myers Squibb: Honoraria; SymBio Pharmaceutical: Consultancy, Honoraria; Otsuka Pharmaceutical: Research Funding; Ono Pharmaceutical: Research Funding; Shionogi: Research Funding; Suitomo Dainippon Pharma: Research Funding; Taiho Pharmaceutical: Research Funding; Teijin: Research Funding; Pfizer Japan: Research Funding; Mochida Pharmaceutical: Research Funding; Yakult Honsya: Research Funding; Perseus Proteomics: Research Funding. von Tresckow: Pentixafarm: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; AstraZeneca: Honoraria, Other: congress and travel support; Amgen: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Other, Research Funding; BMS-Celgene: Consultancy, Honoraria, Other: congress and travel support; Novartis: Consultancy, Honoraria, Other: congress and travel support, Research Funding; Kite-Gilead: Consultancy, Honoraria; MSD: Consultancy, Honoraria, Other: congress and travel support, Research Funding; AbbVie: Other: congress and travel support. Shipp: Bristol Myers Squibb: Research Funding; Merck: Research Funding; AstraZeneca: Consultancy, Research Funding; Immunitas Therapeutics: Consultancy; Abbvie: Other: Institution: Research Grant/Funding; Bayer: Other: Institution: Research Grant/Funding. Herrera: Genentech: Consultancy, Research Funding; Karyopharm: Consultancy; Seagen: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Takeda: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding; Gilead Sciences: Research Funding; Merck: Consultancy, Research Funding; Tubulis: Consultancy. Lin: Merck (MSD): Current Employment. Kim: Merck: Current Employment, Other: Current Stockholder. Chakraborty: Merck & Co., Inc.: Current Employment, Other: Current Stockholder. Marinello: Merck & Co., Inc.: Current Employment, Other: Current Stockholder. Moskowitz: Merck & Co., Inc.: Research Funding.

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Abstract Background The current susceptibility breakpoint for MIN against STM is 4mg/L, yielding &gt;99% of isolates susceptible. Unfortunately, there are limited pre-clinical and clinical data to support this breakpoint for STM. The purpose of this study was to evaluate the efficacy of a MIN human simulated regimen (HSR) against STM across a wide range of MICs in the murine neutropenic thigh model. Methods Clinical STM with modal MIN MICS of 0.25-8mg/L were included. Confirmatory pharmaco*kinetic (PK) studies were performed in infected neutropenic mice to develop a MIN HSR providing an area under the curve (AUC) and maximum concentration (Cmax) exposure similar to MIN 100mg intravenous (IV) q12h at steady-state based on PK parameters from critically ill adult patients. The murine neutropenic thigh infection model was utilized to examine the antibacterial effects of the confirmed MIN HSR against 17 STM. Both thighs of neutropenic ICR mice were inoculated with bacterial suspensions of 107 colony forming units (CFU)/mL. Two hours after inoculation, the MIN HSR was administered subcutaneously (SC) over 24h. Control mice received normal saline. Efficacy was measured as the change in log10CFU/thigh at 24h compared with 0h controls. Results MIN 22, 10, 14, and 10mg/kg dosed SC at 0, 6, 12, and 18h best recapitulated the human Cmax and AUC profile. Mean ± standard deviation bacterial burden at 0h across all isolates was 6.03±0.32 log10CFU/thigh. Bacterial growth was 1.35±0.68 log10CFU/thigh in 24h controls. Six of 7 isolates (86%) with MIC ≤ 0.5mg/L achieved 1-log kill with MIN HSR (-1.44±1.37 log10CFU/thigh). All STM with MIC ≥ 1mg/L experienced bacterial growth (1.18±0.79 log10CFU/thigh) (Figure). Figure. Efficacy of a minocycline human simulated exposure of 100mg intravenous Q12h in the murine neutropenic thigh model against 17 clinical Stenotrophom*onas maltophilia isolates Conclusion These data describe the in vivo efficacy of a MIN HSR with exposures similar to MIN 100mg IV q12h in critically ill adults. Lack of antibacterial activity against STM with MICs ≥ 1mg/L justifies a reassessment of the current susceptibility breakpoint. The study was funded under FDA Contract 75F40120C00171. Disclosures David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase (Other Financial or Material Support, I have been a consultant, speakers bureau member, or have received research funding from the above listed companies.) Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)BioMérieux (Consultant, Research Grant or Support, Speaker’s Bureau)Contrafect (Scientific Research Study Investigator)GSK (Consultant)Merck (Research Grant or Support)Paratek (Speaker’s Bureau)Roche Diagnostics (Research Grant or Support)Shionogi (Research Grant or Support)Summit (Scientific Research Study Investigator)

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Abstract Introduction: Venetoclax (Ven), an oral BCL2 inhibitor, is approved for the treatment (tx) of relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Ven is generally well tolerated, and side effects observed in clinical trials have been consistent with other CLL tx. Clinical trials using the approved dose escalation schedule report negligible rates of clinical tumor lysis syndrome (TLS). We aimed to understand rates of select adverse events (AEs) including cytopenias, infections, and TLS in CLL patients (pts) treated with Ven in community and academic settings. To do so, we examined 297 pts with CLL who received Ven, either alone or paired, in this multicenter, international study. Methods: We conducted a retrospective cohort study of Ven treated pts with CLL across at 15 academic (n=169) and 51 community (n=128) centers outside of the clinical trial setting. This study represents a collaboration between US centers, CLL Collaborative Study of Real World Evidence (CORE), and UK CLL Forum. Demographics, baseline disease characteristics, Ven dosing, TLS risk (per FDA Ven label) and prophylaxis, and AEs were collected. Lab vs. clinical TLS was defined by Howard criteria. PFS was estimated by Kaplan Meier methodology. All comparisons were descriptive. Results: Of the 297 pts examined, median age at Ven initiation was 67 (range 37-91). The group was 69% male, 96% had R/R CLL, and 45% had del17p. Baseline characteristics stratified by practice setting are included in Table 1. 80% received Ven as monotherapy while 20% received it paired with another agent (anti-CD20 mAb (75%), ibrutinib (8.5%), other (16.5%)). All pts were treated outside of clinical trials. During dose escalation, 81% achieved a 400 mg dose and 65% maintained 400 mg following escalation (cyp3A4 use unknown). TLS risk was low in 40%, intermediate (int) in 32%, and high risk in 28%. CT scan prior to Ven initiation was performed in 62%. At least one hospitalization occurred for 56% of low, 80% of int, and 88% of high risk pts (63% of the total cohort). Table 1 describes the distribution of TLS risk and frequency of hospitalizations in academic, community centers. TLS prophylactic measures were available for a subset of pts. Allopurinol was used for 91% (n=68/75) of low, 93% (n=52/56) of int, and 94% (n=29/31) of pts at high risk for TLS. Rasburicase was used for 27% (n=28/102) of low, 42% (n=34/81) of int, and 72% (n=57/79) of high risk pts. Normal saline was used in 85% (n=62/73) of low, 88% (n=49/56) of int, and 97% (n=30/31) of high risk pts. TLS occurred in 8.4% of pts (n=25/297). Three lab and 2 clinical events occurred in low risk pts, 7 lab and 3 clinical events in int risk pts, and 7 lab and 3 clinical events in high risk pts. Of pts with TLS, 1 has discontinued Ven. Of pts with clinical TLS, all were hospitalized, received allopurinol and normal saline, and 28% received rasburicase. 72% with TLS had creatinine clearance <80 mg/mL vs. 44% who did not have TLS (p=0.02). One int risk pt received hemodialysis. One death from TLS (previously reported) was observed in a pt hospitalized with rapid disease progression and tx-related neutropenia re-challenged with 400 mg Ven after dose interruption without dose escalation. Select AEs were neutropenia (ANC<1000) 39.6%, thrombocytopenia (plt <100) 29.2%, infection 25%, neutropenic fever 7.9%, and diarrhea (>7 stools/day) 6.9%. For the subset who received Ven paired, AEs were not increased: 35% neutropenia, 29% thrombocytopenia, 22% infection, 6.3% neutropenic fever, and 6.4% diarrhea. TLS was observed in 3.4% of pts who received Ven paired vs. 9.3% who received Ven monotherapy. 29% pts required ≥1 dose reduction and 32% had ≥1 dose interruption. Median length of dose interruption was 7 days (range 1 - 132). 22 pts (7.4%) discontinued Ven due to an AE. PFS was similar in pts with ≥1 dose interruption vs. 0, pts who required dose interruption ≥8 days vs. <8 days, and pts who achieved a stable Ven dose of <400 mg vs. 400 mg (Figure 1). Conclusions: Ven was well tolerated in this cohort; AE rates were similar to those reported in clinical trials. Both academic and community sites employed TLS prophylaxis consistent with FDA/EMA recommendations resulting in a small proportion of clinical TLS events (<3%). Of note, Ven paired with another agent did not appear to result in increased rates of AEs and TLS events. Dose reduction and interruptions were consistent with clinical experience with other novel agents though these did not appear to impact PFS. Disclosures Fox: Sunesis: Consultancy; Celgene: Consultancy, Other: Travel support, Speakers Bureau; Roche: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Personal fees and non-financial support, Speakers Bureau. Eyre:Gilead: Consultancy, Other: travel support; Abbvie: Consultancy, Other: travel support; Roche: Consultancy; Janssen: Consultancy, Other: travel support; Celgene: Other: travel support. Allan:Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees. Schuster:OncLive: Honoraria; Physician's Education Source, LLC: Honoraria; Dava Oncology: Consultancy, Honoraria; Genentech: Honoraria, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Nabhan:Cardinal Health: Employment, Equity Ownership. Hill:Amgen: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Shah:Lentigen Technology: Research Funding; Miltenyi: Other: Travel funding, Research Funding; Juno Pharmaceuticals: Honoraria; Exelexis: Equity Ownership; Oncosec: Equity Ownership; Geron: Equity Ownership. Lamanna:Jannsen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cheson:AbbVie, Roche/Genentech, Pharmacyclics, Acerta, TG Therapeutics: Consultancy. Coombs:AROG: Other: Travel fees; H3 Biomedicine: Honoraria; Abbvie: Consultancy; DAVA Oncology: Honoraria; Incyte: Other: Travel fees. Barr:AbbVie, Gilead: Consultancy. Skarbnik:Gilead Sciences: Honoraria, Speakers Bureau; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Shadman:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; AstraZeneca: Consultancy; Genentech: Consultancy; Celgene: Research Funding; Verastem: Consultancy; Gilead Sciences: Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy; Genentech: Research Funding; Mustang Biopharma: Research Funding; Beigene: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy. Ujjani:AbbVie: Consultancy, Speakers Bureau. Pagel:Pharmacyclics, an AbbVie Company: Consultancy; Gilead: Consultancy. Jacobs:Genentech: Honoraria. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria. Brander:Genentech: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Novartis: Consultancy, Other: DSMB; BeiGene: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; DTRM: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Acerta: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; AbbVie: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria. Mato:Pharmacyclics: Consultancy, Honoraria, Research Funding; TG Therapeutics: Research Funding; Regeneron: Research Funding; Sunesis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Acerta: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; Prime Oncology: Speakers Bureau.

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Abstract Background: PTHrP-secreting pancreatic neuroendocrine tumors (PNET) are a recognized cause of malignancy associated hypercalcemia. Herein, we report a case of severe hypercalcemia due to an extreme elevation of PTHrP from a PNET, where after treatment of the hypercalcemia, symptomatic hypocalcemia occurred. Clinical Case: A 59 year-old-woman with a recurrent PNET with liver metastases undergoing an evaluation for multi-visceral transplant presented with acute confusion, nausea and vomiting. Diagnostic testing identified an extreme elevation of total calcium (Ca) [&gt;20.1 mg/dL (8.5 - 10.2)] from two different samples [serum albumin 4.1 g/dL (3.9 - 4.9)]. The total Ca level one month earlier was 8.3 mg/dL with a serum albumin of 3.1 g/dL. Total Ca measurements were performed with the Ca Gen.2 assay on a cobas c702 chemistry analyzer (Roche Diagnostics). Results greater than the analytical measurement range (0.8 – 20.1 mg/dL) were diluted with saline and confirmed (22.6 mg/dL). A Radiometer ABL 800 Flex blood gas analyzer was used to determine the ionized Ca concentration [2.94 mmol/L (1.08 - 1.30)]. Upon presentation the serum creatinine (Cr) was 2.07 mg/dL (0.58 - 0.96); eGFR utilizing the MDRD equation 24 mL/min/1.73m2; baseline serum Cr 0.78 mg/dL. Her serum 25-OH vitamin D was 31 ng/mL (31.0 - 80.0), PTH 12 pg/mL (15 - 65), phosphate 4.3 mg/dL (2.7 - 4.8) and 1, 25-OH vitamin D 39.1 (15.0 - 60.0). PTHrP measurements were performed by ARUP Laboratories via liquid chromatography tandem mass spectrometry (LC-MS/MS) and resulted in a reported value of &gt;2500 pmol/L (0.0 - 3.4). Her symptoms resolved and the corrected Ca gradually decreased to 8 mg/dL after treatment with IV fluids, calcitonin 200 units sc every 12 hours for 48 hours, 60 mg IV pamidronate, and five sessions of hemodialysis. Within thirteen days of receiving pamidronate, her corrected Ca slowly increased to 12mg/dL; thus, she received a single dose of 120 mg sc denosumab. Nine days later, the patient developed symptomatic hypocalcemia (7.3 mg/dL) manifested by paresthesia in the hands and feet and perioral numbness. She then received multiple doses of oral and intravenous Ca along with 50,000 units of oral ergocalciferol twice weekly. The corrected Ca normalized (8.1 mg/dL) and symptoms resolved. The patient was discharged with plans for future treatment of her underlying malignancy. Conclusion: This is the first report of a PNET producing an extreme elevation of PTHrP of higher than 2500 pmol/L, resulting in a concordant extreme elevation of total calcium within a month of documented normocalcemia. Treatment of hypercalcemia with denosumab may result in the development of hypocalcemia requiring treatment.

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Abstract Coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has exposed clinical laboratories to unprecedented challenges. With surging case numbers, clinical laboratories were forced to acquiesce and integrate multiple testing platforms with varying workflows and analytical sensitivities in order to meet testing volumes. Now a new challenge has emerged with the evolution of viral variants, both globally and locally, raising concerns for uncontrolled spread, increased disease severity, and weakened responses to vaccinations. Preliminary data suggests that these variants may be associated with higher viral titers and prolonged infections. While primarily leveraged for epidemiologic surveillance, the clinical utility of variant detection may quickly become paramount. Furthermore, laboratories must remain vigilant and nimble enough to pivot should variant identification play a role in the patient care. To prepare for the validation of clinical assays that identify important viral variants, we designed a novel method, termed VariantDirect, to screen SARS-CoV-2 positive samples for the presence of variants, focusing initially on the increasingly prevalent UK and South African (SA) variants. The detection strategy is based on primers designed to specifically target the viral receptor-binding domain mutation, N501Y, shared by the UK and SA strains. Screening for variants will be limited to nasopharyngeal swab samples of high viral titers (Ct values &lt;25 by RT-qPCR assay, Roche Diagnostics). Pools of 9 different samples, 50 µl each, are mixed and stored at -80°C along with aliquots of the 9 original samples. These pools will then be tested, and if positive for the N501Y variant, the pooled 9 samples will be thawed and tested separately to identify the affected specimen. Most of these specimens are also being independently sequenced via a comprehensive but more resource-intensive NGS approach. Advantages of our pooled workflow are primarily in time and cost, with the capacity of screening up to 837 specimens on a single run. In addition, our collection strategy establishes a “time capsule” to document the evolution of viral strains within our geographical region. Finally, these studies serve to optimize technical parameters for the development of clinical assays. A validated nucleic acid (NA) extraction-free RT-qPCR method will be utilized for this assay. Our internal validation data showed comparable analytical sensitivities to NA extraction-based methods. Pooled samples in transport medium are diluted in normal saline at a ratio of 1:1, and then heat-inactivated in the presence of proteinase-K and ultimately analyzed on the Applied Biosystems™ 7500 Fast Dx instrument. As new variants of interest emerge, primers and probes can be quickly redesigned and validated on clinical samples within our NGS-confirmed “time capsule”. This study will provide important information needed for current or future genomic and epidemiologic studies.

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PPARα and c-Fos are involved in regulation of gene expression and are known to be dependent on retinoic acid (RA), which in turn influences oocyte growth and developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003. Reproduction 125, 409–416), probably acting in part through granulosa cells. Peroxisome proliferator-activated receptor-α (PPARα) heterodimerizes with the retinoid receptor X (RXR), while c-Jun/c-Fos heterodimerizes with liganded retinoic acid receptors (RARs), then preventing formation of transcription factor activator protein 1 (AP-1) complexes capable of DNA binding. Cellular retinoic acid binding protein (CRABP) limits RA excess and regulates the transcriptional potential of RA;; CRABPII has been detected in rat granulosa cells from mature follicles and luteal cells. The aim of this study was to investigate PPARα, c-Fos and CRABPII mRNA expression in bovine granulosa cells. In parallel, other genes whose expression can be influenced by RA were analyzed: luteinizing hormone receptor (LHr), follicle stimulating hormone receptor (FSHr), aromatase and growth hormone (GH). Ovaries were collected at a local abattoir and kept in saline at 30–35°C. Granulosa cells were obtained by aspirating 2- to 7-mm antral follicle contents, pelleted at 700g for 4min and resuspended in RNA-later (Ambion®). Total RNA was isolated with a NucleoSpin® RNAII kit (Macherey-Nagel), and mRNA was reverse transcribed into single-stranded cDNA using a 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche). A PCR standard method was made using 1μL of the cDNA as a template. All PCR primer couples were designed on the basis of the bovine sequence, but c-Fos and CRABPII primers were designed based on the human-murine sequences. Primers within the couple were located in different exons to distinguish DNA from RNA amplification. CRABPII was further investigated in bovine whole ovary, corpus luteum (CL) and liver, in a search for positive controls. Bovine β-actin, 18S and 28S were examined in each sample as positive controls for RNA isolation and cDNA synthesis efficiency. TenμL of product were loaded into an agarose 2% gel in TBE buffer containing ethidium bromide, and were separated by horizontal electrophoresis. Gels were visualized with ultraviolet light and photographed using a digital camera. Gene expression in granulosa was demonstrated for PPARα, c-Fos, LHr, FSHr, aromatase, GH and controls (β-actin, 18S and 28S) but CRABPII gene did not express in granulosa cells, whole ovary, CL or liver under our experimental conditions. While lacking CRABPII expression remains intriguing, the expressed genes support a role of retinoid pathway within granulosa cells under both in vivo and in vitro conditions, because granulosa cells used in the present experiments were derived from follicles providing oocytes for IVM-IVF. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

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Abstract Pre-analytical error may occur when collecting blood samples from intravenous (IV) lines. Blood samples can be inadvertently diluted by exogenous solutions if an insufficient amount of blood is discarded prior to collection. Rules built in the laboratory information system or middleware can be used to alert laboratory staff to specimens that require co-investigation with the clinical team to determine if sample dilution is possible or likely. Currently, however, there is a lack of literature to guide laboratorians on how to define quality rules of this nature, particularly for detecting subtle, rather than gross contamination by commonly administered IV-fluids. Accordingly, the primary objective of this study is to derive sensitive and specific, multivariate delta checks to detect more subtle blood sample contamination by commonly used crystalloid solutions. To derive the rules, we began with in vitro experiments by spiking increasing volumes of major IV-fluids (normal saline (NS), lactated ringers (LR), and 5% dextrose (D5W)) into clinical blood samples that were collected from healthy donors (n=3). Crystalloid solutions were serially spiked into blood samples at 10% (solution:sample) increments for NS and LR from 10% up to 90%, and in 5% increments for D5W, up to 50%. Basic metabolic panel (BMP) analytes were measured and compared between neat and contrived samples. All testing was performed on Roche Cobas 8000 chemistry auto-analyzers. Based on in vitro data, we derived multivariate delta checks using analytes in a BMP that would reflect 10-40% contamination by a given fluid. We then performed a retrospective data analysis on more than 28 thousand, serially collected BMP results to identify samples that were flagged by these rules. Chart review was then performed to identify EHR-based evidence of temporally associated administration of crystalloid solutions through a peripheral IV access line. Increasing sample dilution from the in vitro study showed significant changes in several BMP analytes across all fluid types. With respect to NS, the most significant changes relative to baseline were observed with potassium, calcium, and chloride, while for LR, the most significant changes relative to baseline were observed with glucose, bicarbonate, and calcium. For D5W, significant changes were observed with glucose, calcium, chloride and sodium. Accordingly, delta check rules were implemented using relative changes of the aforementioned analytes. On chart review, we found 80-100% of flagged samples came from patients with administration of that given fluid in the last 12-24 hours. The results from this study support delta checks that consider multiple analytes, and fluid-specific bias profiles, as this may provide a more sensitive and specific approach to detecting contamination of clinical blood samples by crystalloid solutions. Further evaluation using retrospective data analysis will be used to validate these rules sensitivity and specificity followed by prospective testing before implementation.

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BackgroundPanama’s prevalence of rheumatoid arthritis lies within the global range (.24 to 1%). To improve the quality of life of RMD patients in the country, FUNARP was created over 18 years ago to educate and support patients, caregivers, and civil.FUNARP decided to focus its efforts on digital campaigns since 2020 taking advantage of its coverage, low costs, and ease of usage. Further, the mobile penetration of Panama is 136% of its population [1] making social media the best distribution channel for this purpose.ObjectivesTo expand educational coverage, especially for those with limited mobility or living in hard-to-reach areas. Another objective is to measure the effectiveness of digital media campaigns (paid vs organic growth), as well as to identify the most effective social network for these purposes.MethodsSocial media campaigns complement FUNARP’s annual education plan which consists of monthly onsite and virtual lectures/seminars. The educational approach is based on the physician’s perspective and the patient’s experience. The campaigns consist of a mixed scheme: selected ad investment and in-house content creation; and full-fledged campaigns with professional content development and paid ads. In 2022, a total of five campaigns were launched following this process: a) selection of the campaign theme, b) professional advice on content, c) sponsorship, d) hiring of the marketing agency, e) approval of design and publications, and f) analysis of results to the board of directors and sponsors.Results“Early Diagnosis and Motivation to Consult a Rheumatology Specialist” was the most successful campaign - developed by a marketing agency with videos supporting the message, and paid ads on both Instagram and Facebook, see Figure 1. Salient results: 110,488 accounts reached and 25,975 interactions, see Table 1.Figure 1.Best performing campaign.Campaigns with professional content creation and paid ads in social media ads had more than 50% reach and 90% interaction than the ones without them. Videos have a higher acceptance vs. static images, see Table 1:Table 1.Salient KPI of social media campaigns in 2022 (reach vs. engagement)Qualitative results have been observed by rheumatologists who have indicated that patients who have had access to information from our foundation are more informed and involved in their treatment. They perceive that these patients have a better quality of life compared to those who do not have access to information about their pathology.ConclusionWorking with marketing companies, and rheumatologists, as well as investing in advertising directly influences the effectiveness of health awareness and literacy campaigns. Publications where real patients appear as protagonists had higher acceptance vs. those with stack images and videos, or models. Instagram appears to have a better impact than Facebook.The early diagnosis campaign managed to overcrowd first-time consultations with rheumatologists in the public health system (less than 20 rheumatologists for a 4.2 MM population in Panama). FUNARP observed an increase in requests for information and orientation in the navigability of the system to obtain care with rheumatologists and advice on various health issues from the community through social media messaging services.FUNARP will continue to conduct digital campaigns to expand the educational coverage of the Panamanian population. It is recommended to have advertising investments of more than U.S.$100.00 per publication of each campaign and to have content generated by specialists and advertising agencies. In addition, sponsorship will continue to be sought to increase literacy in patients with RMD.For the next campaigns, the WhatsApp button will be made available to easily address requests and questions from the community. A marketing and advertising commission has been created within FUNARP to keep more detailed metrics and measure other variables in the campaigns that have not been measured so far.Reference[1]Panamanian Public Services Authority 2022Acknowledgements:NIL.Disclosure of InterestsEnma Pinzon Grant/research support from: Abbvie, Roche, Sandoz, Luris Higuera: None declared.

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Expression of some X-chromosome linked genes has recently been shown to be altered in bovine somatic cell nuclear transfer (SCNT) derived embryos (Wrenzycki et al. 2002 Biol. Reprod. 66, 127), implying that the regulatory mechanisms of X-linked transcription are affected by embryo in vitro production (IVP) methods. We analyzed the transcriptional pattern of X-linked genes (BIRC4, GAB3, HPRT1, MECP2, RPS4X, SLC25A6, and XIST) in bovine in vitro fertilized (IVF) and SCNT male and female blastocysts to determine X-inactivation status and changes resulting from IVP. We collected pools of male (n = 5 pools) and female (n = 3 pools) IVF-derived blastocysts (Bousquet et al. 1999 Theriogenology 51, 59) and male (n = 5 pools) and female (n = 3 pools) SCNT-derived blastocysts (Mastromonaco et al. 2004 Reprod. Domest. Anim. 39, 462). Each pool consisted of five blastocysts. Embryos were washed in phosphate buffered saline (PBS) + 0.1% polyvinyl alcohol (PVA), collected, and stored at -80�C. Total RNA was extracted with an Absolutely RNA Microprep kit (Stratagene, La Jolla, CA, USA), DNase I treated, and precipitated with isopropanol and linear acrylamide (Ambion, Inc., Austin, TX, USA) as a carrier. Reverse transcription was performed with Oligo-dT (Invitrogen, Burlington, Ontario, Canada) and Superscript II RT (Invitrogen). Transcript quantification was performed by quantitative real-time PCR using SYBR Green I (LightCycler system, Roche, Diagnostics, Laval, Quebec, Canada). Data analysis was performed with SAS (SAS Institute, Inc., Cary, SC, USA) using a mixed-model factorial ANOVA and with results presented as estimates of the median, ratios of estimates, and 95% confidence intervals with � = 0.05. IVF-derived male and female blastocysts possessed similar levels of the transcripts analyzed, suggesting successful dosage compensation at this developmental stage for embryos fertilized in vitro. XIST was not detected in male IVF embryos. GAB3 was not detected in any of the female groups and, in addition, HPRT1 transcripts were not detected in SCNT derived female embryos. Male and female SCNT-derived blastocysts possessed marked differences in their transcript levels, with males showing statistically significantly higher levels of BIRC4 and RPS4X and females possessing higher levels of MECP2 and SLC25A6 transcripts although differences between the latter two were not statistically significant. XIST was detected in both male and female SCNT blastocysts. We conclude that dosage compensation between male and female IVF blastocysts is achieved at this developmental stage for the transcripts examined. However, this pattern was markedly changed in the SCNT group, affecting especially female SCNT blastocysts, suggesting that the regulatory mechanisms of X-inactivation and X-linked gene expression are substantially altered in SCNT embryos probably due to aberrant epigenetic patterns and faulty genome reprogramming. We are currently analyzing X-linked transcription in male and female in vivo-derived blastocysts in order to compare this group with IVP-derived embryos. This work was funded by NSERC, CIHR, and CRC.

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The aim of this study was to compare the differential effects of the addition of IGF-LongR3 or insulin-like growth factor-1 (IGF-1), during in vitro maturation (IVM) of bovine oocytes on embryonic development, by analysing embryonic viability and apoptosis. Ovaries collected at a slaughterhouse were transported in saline solution (NaCl 0.9%) at 38°C. Follicles of 2–8 mm of diameter were aspirated. Only cumulus-oocyte complexes (COC) with hom*ogeneous cytoplasm, surrounded by at least 3 layers of compact cumulus cells were selected in Dulbecco’ modified PBS 0.3% polyvinyl alcohol (PVA) and transferred to TCM HEPES at 38°C. Maturation medium was composed by TCM199, 0.2 mM pyruvate, 1 μg mL–1 FSH, 50 μg mL–1 LH, 100 μg mL–1 streptomycin sulfate, 100 UI mL–1 penicillin, and 85 μg mL–1 amikacin. Four experimental groups were determined using basic medium supplemented with fetal bovine serum (FBS; 10%), PVA (3 mg mL–1 Polyvinylpyrrolidone), IGF-1 (3.100 ng mL–1 insulin growth factor), LongR3-IGF (14.100 ng mL–1 long-chain human insulin growth factor 1-r3) respectively. The IVM was performed in Petri dishes (35 × 15 mm) with 90-μL droplets of the respective media, covered with sterile mineral oil, in 5% CO2 and 38.5°C temperature atmosphere for 22–24 h. The matured oocytes were fertilised with 2 × 106 spermatozoa mL–1 and incubated for 18 h. Embryos were denuded and cultivated in SOFaa medium (2.7 mM myoinositol, 0.2 mM pyruvate, 5 mg mL–1 BSA, 100 μg mL–1 streptomycin, 100 UI mL–1 penicillin, 85 μg mL–1 amikacin, and 25 μL mL–1 of FBS). Six repetitions were performed. After 7 days, blastocyst formation was analysed and all blastocysts were submitted to the TUNEL reaction. (In Situ Cell Death Detection Kit with Fluorescein, Roche®, Mannheim, BW, Germany), according to the technique adapted by Paula-Lopes and Hansen (2002).Green nuclei fluorescence (fluorescein isothiocyanate; FITC) was considered with fragmented DNA. Data were analysed by ANOVA), and least-squares means was used to verify differences of means using the PROC GLIMMIX model of SAS statistical software package (SAS Institute, Cary, NC, USA). Cleavage rate was similar for all groups. There was no statistical difference (P > 0.05) between groups regarding to embryo production. Most of the blastocysts obtained at Day 7 of culture had good quality (grade I). However, a difference (P = 0.03) on the number of expanded blastocyst was found between FBS (20.83 ± 3.22) and PVA (10.18 ± 3.22) groups. Furthermore, IGF-1 (12.03 ± 1.60) group showed a higher (P = 0.04, P = 0.02) apoptosis rates compared to groups FBS (7.96 ± 1.29) and PVA (6.97 ± 1.48). In the present study, it was observed that the addition of IGF-1 or LongR3-IGF during IVM provided a similar embryo production compared to FBS. On the other hand, embryos obtained from oocytes maturated on medium with the addition of IGF-1 had a high number of cells with fragmented DNA, indicating a more apoptosis.

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Background:Cystatins are cysteine proteases-inhibitors secreted byFasciola hepaticain order to modulate the host immune response to promote survival of the parasite. These molecules are able to inhibit different mammal cathepsins, to regulate the immune balance via Th2 and T regulatory responses, to downregulate antigen presentation and the release of pro-inflammatory cytokines (1,2) – mechanisms that are important in the development and maintenance of several immunopathology, as rheumatoid arthritis (RA) (3).Objectives:To evaluate the therapeutic effect of recombinants cystatin 1 and cystatin 3 fromFasciola hepaticain a mice model of collagen-induced arthritis (CIA).Methods:Twenty-seven DBA/1J mice were induced with CIA by an injection of collagen type-II and Freund’s adjuvant at days 0 and 18. Animals were randomly divided into three groups: vehicle (n=9, treated with phosphate-buffered saline), cystatin 1 (n=9, treated with 100 µg/dose of recombinant cystatin 1) and cystatin 3 (n=9, treated with 100 µg/dose of recombinant cystatin 3). Treatment started after day 18 by intraperitoneal injection once a day until the end of the experiment, at day 45 after CIA induction. Clinical arthritis score, nociception, paw edema, body and spleen weight were evaluated. Lymphocytes were isolated from lymph nodes and CD4+CD25+Foxp3+ T regulatory subset was assessed by flow cytometry. Data are expressed as mean ± SEM and were evaluated by one-way or two-way ANOVA followed by Bonferroni post-test.Results:Treatment with cystatin 1 did not alter any of the analyzed parameters. On the other hand, cystatin 3 was able to reduce clinical arthritis score from day 38 with 32% of reduction at day 45 (9.22±1.22) compared to vehicle (13.56±0.73) (p<0.05). In addition, treatment with cystatin 3 diminished nociception (cystatin 3: 4.0±0.36g, vehicle: 2.7±0.32g) (p<0.05) and paw edema (cystatin 3: 0.051±0,012ml, vehicle: 0.093±0.007ml) (p<0.05). Moreover, the treatment did not alter body weight (cystatin 3: 21.67±0.31g, vehicle: 21.05±0.38g) and spleen weight (cystatin 3: 7.04±0.31, vehicle: 7.16±0.38), as well as the T regulatory population (cystatin 3: 63.38±3.66%; vehicle: 58.31±6.77%).Conclusion:Treatment with cystatin 3 improved collagen-induced arthritis by attenuating the disease score, nociception and paw edema. Moreover, the treatment did not induce body weight loss or spleen weight alteration. These results suggest that recombinant cystatin 3 fromFasciola hepaticahas the potential as a treatment for inflammatory and autoimmune diseases such as RA.References:[1]Klotz C, Ziegler T, Daniłowicz-Luebert E, Hartmann S. Cystatins of parasitic organisms. Adv Exp Med Biol. 2011;712:208-21.[2]Vray B, Hartmann S, Hoebeke J. Immunomodulatory properties of cystatins. Cell Mol Life Sci. 2002 Sep;59(9):1503-12.[3]Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet. 2016 Oct 22;388(10055):2023-2038.Disclosure of Interests:Mirian Farinon: None declared, Renata Ternus Pedo: None declared, Thales Hein da Rosa: None declared, Barbara Jonson Bartikoski: None declared, Thaís Karnopp: None declared, Martin Cancela: None declared, Henrique Bunselmeyer Ferreira: None declared, Ricardo Xavier Consultant of: AbbVie, Pfizer, Novartis, Janssen, Eli Lilly, Roche

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Abstract Background: IFN-α2 is indicated for the therapy of hairy cell leukemia, chronic myelogenous leukemia, follicular lymphoma, and malignant melanoma. As is the case for most cytokines, the pharmaco*kinetic (PK) properties of IFN-α2 are critical for dosing and efficacy. In vivo, the protein is quickly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Pegylation of IFN-α2 significantly increases the serum half-life and reduces renal clearance, thus enhancing its efficacy. However, established pegylation of IFN-α2 results in a mixture of positional isomers and reduced in vitro activity, as known for PEGASYS (Roche) and PEG-INTRON (Schering-Plough). Methods: To improve the PK properties and the potency, the DNL method (Rossi et al. Proc. Natl. Acad. Sci. USA, 2006,103:6841) was used to generate novel agents having two copies of IFN-α2b conjugated to polyethylene glycol (PEG). Results: A fusion protein (DDD2-IFN-α2b) composed of IFN-α2b with a dimerization-and-docking domain (DDD2) and a six-His tag was expressed both in myeloma cells and in E. coli. Two PEG-based modules, each composed of a fluorescent molecule, an anchor domain (AD) and either a 20-kDa PEG (IMP362) or a 30-kDa PEG (IMP413), were synthesized. Combining DDD2-IFN-α2b and IMP362 or IMP413 under redox conditions resulted in the desirable DNL conjugates consisting of two copies of IFN-α2b and one PEG linked site-specifically via the DDD and AD interaction. The cytotoxic activity of DDD2-IFN-α2b on Daudi lymphoma cells was similar to that of commercially available recombinant IFN-α2 (rhIFN-α2). The purity and identity of the two DNL conjugates (α2b-413 and α2b-362) were demonstrated by SDS-PAGE, immunoblotting, and fluorescence imaging. Both also showed potent cytotoxic activity on Daudi cells in vitro and superior PK properties to PEG-INTRON. For example, the mean blood residence times for α2b-362 (10.3 h) and α2b-413 (21.7 h) were significantly longer than those of rhIFN-α2 (0.7 h) and PEG-INTRON (5.1 h). Initial studies in mice bearing Daudi xenografts showed a significant therapeutic advantage over PEG-INTRON for both α2b-362 and α2b-413. Animals given 14,000 IU of PEG-INTRON had a median survival (MS) of 32 days compared to 21 days for saline control, whereas those receiving α2b-362 at 14,000 IU, 7,000 IU and 3,500 IU resulted in MS of 45, 41 and 32 days, respectively. More remarkably, α2b-413 was the most effective, achieving MS of 46, >53, and >53 days with 3,500 IU, 7,000 IU and 14,000 IU, respectively, all statistically significant improvements (P< 0.0028) compared to PEG-INTRON given at each equivalent activity. Conclusions: The DNL method provides a novel pegylation strategy for generating a dimeric IFN-α2b that is linked site-specifically to a single PEG at the predetermined location. Since the resulting conjugates exhibit improved PK and efficacy in a Burkitt lymphoma model, they may represent a new class of interferons for use in cancer and infectious disease therapy.

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Background Primary biliary cirrhosis (PBC) is an autoimmune liver disease, and 50% of patients with this disease experience fatigue. This is a debilitating symptom affecting quality of life and resulting in social isolation, which is highlighted by patients as a research priority. PBC is characterised immunologically by the presence of high-titre autoantibodies that are directed at the pyruvate dehydrogenase complex (PDC) and are highly effective at blocking its energy generation function. We hypothesised that if anti-PDC antibodies were a driver of fatigue through bioenergetic dysfunction, then the B-cell-targeting biological agent rituximab (MabThera®, Roche Products Ltd, Welwyn Garden City, UK) might be a therapeutic option. Objective To assess whether or not rituximab safely improved moderate or severe fatigue in PBC patients. Design A Phase II, double-blind, randomised controlled trial comparing rituximab with placebo in fatigued PBC patients. Randomisation was conducted using a web-based system. Participants received two infusions on days 1 and 15 and were followed up at 3, 6, 9 and 12 months. Setting A single-centre UK study in Newcastle upon Tyne Hospitals NHS Foundation Trust. Participants Seventy-one participants aged ≥ 18 years with PBC and moderate or severe fatigue (score of > 33 on the PBC-40 fatigue domain) were screened. The PBC-40 questionnaire is a fully validated disease-specific health-related quality-of-life measure for use in patients with PBC. Fatigue, with a maximum score of 55, is one of its six domains. Fifty-seven participants were randomised to the trial, 55 of whom reached the primary end-point assessment. Intervention Participants were randomised in a 1 : 1 ratio to receive either rituximab (1000 mg) or a saline intravenous infusion (placebo) on days 1 and 15. The infusions were delivered in a double-blind manner using the same protocol. Main outcome measures The primary outcome measure was the PBC-40 fatigue domain at 3 months, assessed on an intention-to-treat basis. Secondary outcome measures included markers of bioenergetics function (anaerobic threshold and post-exercise muscle pH assessed using magnetic resonance imaging) and physical activity levels. Impact on biochemical markers of liver disease severity was assessed as an experimental outcome. Results Rituximab therapy was safe, with no serious adverse events linked to the drug. There was no statistically significant difference in fatigue score at 3 months between the rituximab and placebo arms [adjusted mean difference –0.9, 95% confidence interval (CI) –4.6 to 3.1]. However, improvement in fatigue was observed in both arms {mean score decreasing from 41.2 [standard deviation (SD) 5.5] to 36.2 (SD 8.4) in the rituximab arm and from 43.0 (SD 5.9) to 38.1 (SD 8.7) in the placebo arm}. There was little difference in any of the secondary outcomes between arms. However, anaerobic threshold improved significantly in the rituximab arm (adjusted mean difference at 3 months 1.41, 95% CI 0.03 to 2.80). No change in muscle bioenergetics characteristics was seen. A suggestive improvement in liver biochemistry was observed. Limitations Recruitment was lower than the original target, leading to a reduction in study power. A clinically significant placebo effect on PBC-40 fatigue scores was seen. Conclusions Rituximab is ineffective for the treatment of fatigue in unselected PBC patients despite metabolic modulation through improvement of anaerobic threshold. Future work Results from the trial demonstrate that metabolic effect of rituximab is not translated into clinical benefit. This will help to guide us to design future trials and when looking at completely different targets. Trial registration Current Controlled Trials ISRCTN03978701, ClinicalTrials.gov identifier NCT02376335 and EudraCT number 2012-000145-12. Funding This project was funded by the National Institute for Health Research (NIHR) Efficacy and Mechanism Evaluation programme and will be published in full in Efficacy and Mechanism Evaluation; Vol. 5, No. 2. See the NIHR Journals Library website for further project information. Additional funding was received from the Medical Research Council and a Department of Health and Social Care subvention.

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Abstract Abstract 4801 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic and leukemic cells. Reactive oxygen species (ROS) level is highly regulated. This regulation involves antioxydative enzymes and it has been recently described that leukemic stem cells (LSC) overexpress glutathione peroxydase 3 (Herault O et al, J. Exp. Med, 2012). This overexpression is associated with a decrease in ROS level and p38MAPK inactivation. ROS level in leukemic cells could be also regulated by the activity of ROS producers, such as NADPH oxidase, known to catalyze an electron transfer from NADPH to oxygen producing superoxides which could generate other downstream ROS. The expression of this enzymatic complex (NOX family, 6 isoforms) has been established in the plasma cell membrane of normal CD34+ hematopoietic progenitors (Piccoli C et al, Biochem. Biophys. Res. Commun., 2007). The aim of this study was to decipher the expression of NADPH oxydase components in various human acute myeloid leukemia (AML) Different leukemic cell lines were used according FAB classification: KG1a (MO/M1), KG1 (M1), HL60 (M2), Kasumi 1 (M2), NB4 (M3), ML2 (M4), THP1 (M5), U937 (M5), MV4–11 (M5), K562 (M6). The cells were cultured (2.105 cells/mL, 37°C in 95% humidified air and 5% CO2) in RPMI 1640 with 20mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100mg/mL streptomycin. The expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2, P22phox and P40phox, P47phox, P67phox, NOXO1, NOXA1 was quantified by RT-qPCR (Universal Probe Library, Roche). NOX2 and its regulatory subunits expression was quantified by SDS-PAGE and western-blot experiments. The effects of diphenylene iodonium (DPI), a specific NOX inhibitor, were evaluated by ROS quantification using dichlorodihydrofluorescein diacetate (DCF-DA) staining followed by fluorimetry and flow cytometry analyses. The cells were washed twice in the physiological saline buffer (PBS) without calcium and magnesium, then incubated in PBS complemented with 0.5M MgCl2, 0.9M CaCl2, 20mM glucose (Picciocchi A et al, J. Biol. Chem., 2011) with or without 20μM DPI for 1 hour. The cells were distributed at 106cells per 200μL well in 96 wells plates. DCF-DA (10μM) was added to quantify the ROS level (flow cytometry) and to monitor ROS production kinetic (fluorimetry). NOX family genes expression showed that phagocyte oxidase NOX2 is expressed in all leukemic cell lines. Conversely the NOX2 isoforms were not expressed, or very weakly expressed in leukemic cell lines (NOX3 in KG1a; NOX4 in K562; DUOX1 in KG1a, KG1; DUOX2 in KG1a, KG1, HL60). P22phox, the second cytochrome b558 component was also expressed in all cell lines, this expression being higher than NOX2. The cytochrome b558 components were more expressed in differentiated leukemic cells (granulocytic and monocytic) than in undifferentiated cells (KG1a, KG1). NOX2 regulatory subunits were expressed in all leukemic cell lines, the lower level (especially P40phox, P47phox) being observed in KG1a. Proteins quantification confirmed RNA results. Cytochrome b558 components and regulatory subunits were expressed in all cell lines with a higher level in differentiated leukemias. Interestingly, the regulatory subunits were not observed in KG1a cells. Functional flow cytometry and fluorimetry studies revealed a decrease in ROS production in DPI exposed leukemic cell lines. This effect was higher in monocytic cell lines than in granulocytic and undifferentiated leukemias. In conclusion, NADPH oxidases are present in the AML cell membrane, and NOX contribution to the ROS level is higher in differentiated cells than in immature leukemias. Altogether these results suggest that NADPH oxidase is constitutively active in leukemic cells and influences the ROS level, suggesting a role in the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

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Abstract Background We showed earlier that prolonged treatment with a combination of azacitidine (DNMT1 inhibitor) and panobinostat (HDAC inhibitor) induced complete remission in a disseminated xenograft model of KMT2A rearranged AML (Gopalakrishnapillai et al., 2017, Leuk Res). Pretreatment with epigenetic drugs has been shown to induce chemo-sensitivity in some malignancies. However, studies on the use of epigenetic drugs in overcoming chemoresistance mediated by the bone marrow microenvironment are limited. The aim of this study was to determine the merit of incorporating epigenetic therapy with chemotherapy in AML treatment regimen and to identify the mechanism by which epigenetic drugs can chemosensitize AML. Methods AML cell lines and PDX lines were co-cultured with HS5 bone marrow stromal cells. Cell viability following drug treatment was assessed by flow cytometry. For determination of cell adhesion, violet proliferation dye stained AML cells were co-cultured with HS5 cells. Following treatment, non-adherent cells were removed by washes with phosphate buffered saline. The number of adherent AML cells was determined by flow cytometry. NSG-B2m mice were engrafted with MV4;11 cells and bled regularly to evaluate disease progression. Once engraftment was confirmed mice were assigned to treatment groups. Epigenetic therapy constituted azacitidine and panobinostat (2.5 mg/Kg each; Qd5). Chemotherapy constituted cytarabine (50 mg/Kg; Qd5) and daunorubicin (1.5 mg/Kg; Qd3). Mice were euthanized when they reached predetermined experimental endpoints. All animal studies were approved by the Institutional Animal Care and Use Committee. Results AML cells in co-culture with HS5 bone marrow stromal cells were less sensitive to chemotherapeutics cytarabine and daunorubicin compared to AML cells in monoculture. This chemoprotection was not achieved when AML cells were cultured in HS5 conditioned media or in Transwell inserts suspended over HS5 monolayers, suggesting that direct cell-to-cell contact was required. MV4;11 cells exposed to epigenetic drugs or cytarabine alone retained 70% viability. Pre-treatment with the epigenetic drug combination of azacitidine and panobinostat prior to cytarabine exposure greatly reduced cell viability in MV4;11 cells harboring KMT2A fusion (Fig. 1A). Similar chemo-sensitization was observed when four distinct KMT2A rearranged PDX lines were used ex vivo. This sensitization was accompanied by reduced binding of AML cells to HS5 cells following treatment with epigenetic drugs (Fig. 1B). We evaluated the efficacy of the epigenetic therapy and chemotherapy combination in disseminated xenograft models of pediatric AML. NSG-B2m mice transplanted with MV4;11 cells via the tail vein were randomly assigned to four groups - 1) vehicle, 2) epigenetic therapy 3) chemotherapy (cytarabine + daunorubicin), and 4) epigenetic therapy followed by chemotherapy. The mice receiving the epigenetic therapy and chemotherapy combination survived significantly longer than mice treated with any other condition (Fig. 1C). To assess the leukemic cell distribution in peripheral blood, bone marrow and spleen following treatment, a cohort of mice receiving epigenetic therapy alone or chemotherapy alone were euthanized a day after treatment concluded. The percentage of leukemic cells in bone marrow and spleen was lower in mice treated with epigenetic therapy than those treated with chemotherapy, consistent with the survival data. Surprisingly, the peripheral blood counts were significantly higher in mice receiving epigenetic drug combination. These results together with our in vitro data indicate that epigenetic therapy induces mobilization of AML cells to the blood stream. This increased availability of AML cells may promote enhanced sensitivity to chemotherapy. Conclusion Our data suggest that direct cell-to-cell contact plays a major role in mediating chemoprotective effects of the bone marrow microenvironment. These chemoprotective effects can be overcome by pretreatment with epigenetic drug combination azacitidine and panobinostat. Epigenetic drugs interfere with AML cell interactions with the microenvironment and dislodge AML cells from their protective niche. Thus, mobilization of AML cells to peripheral blood is a potential mechanism of chemo-sensitization mediated by epigenetic drugs. Disclosures Kolb: Servier: Membership on an entity's Board of Directors or advisory committees; Roche- Genentech: Membership on an entity's Board of Directors or advisory committees.