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An association between certain ABO/Rh blood groups and susceptibility to SARS-CoV-2 infection has been proposed for adults, although this remains controversial. In children and adolescents, the relationship is unclear due to a lack of robust data. Here, we investigated the association of ABO/Rh blood groups and SARS-CoV-2 in a multi-center study comprising 163 households with 281 children and 355 adults and at least one SARS-CoV-2 seropositive individual as determined by three independent assays as a proxy for previous infection. In line with previous findings, we found a higher frequency of blood group A (+ 6%) and a lower frequency of blood group O (-6%) among the SARS-CoV-2 seropositive adults compared to the seronegative ones. This trend was not seen in children. In contrast, SARS-CoV-2 seropositive children had a significantly lower frequency of Rh-positive blood groups. ABO compatibility did not seem to play a role in SARS-CoV-2 transmission within the families. A correction for family clusters was performed and estimated fixed effects of the blood group on the risk of SARS-CoV-2 seropositivity and symptomatic infection were determined. Although we found a different distribution of blood groups in seropositive individuals compared to the reference population, the risk of SARS-CoV-2 seropositivity or symptomatic infection was not increased in children or in adults with blood group A or AB versus O or B. Increasing age was the only parameter positively correlating with the risk of SARS-CoV-2 infection. In conclusion, specific ABO/Rh blood groups and ABO compatibility appear not to predispose for SARS-CoV-2 susceptibility in children.
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BACKGROUND: Capillary sampling of blood counts is a well-established alternative to venipuncture in paediatrics. However, the sampling method has to be considered when interpreting test results, as measurements differ. Ethical and practical considerations prevent simultaneous venous and capillary sample acquisition in comprehensive paediatric cohorts that span all ages for the purpose of a direct method comparison, resulting in uncertainty regarding the interpretation of capillary test results. METHODS: We applied a data mining method to calculate the differences between capillary and venous blood count analytes using laboratory data collected during patient care. We examined 486 401 blood counts performed between 2010 and 2017 in two German paediatric tertiary care centers in children from birth to 18 years analysed on SYSMEX XE-2100 and SYSMEX XE-5000 devices, and analysed the differences between capillary and venous test results in 15 218 paired samples performed within 24 h. RESULTS: We identified the mean systematic differences between capillary and venous (capillary-venous) test results for haemoglobin (+6.5 g/L), haematocrit (+2.38%), platelet count (-7.01 × 109 /l), red cell count (+0.18 × 1012 /L), white cell count (-0.64 × 109 /L), mean corpuscular cell volume (+2.07 fl), mean corpuscular haemoglobin (+0.33 pg), mean corpuscular haemoglobin concentration (-4.4 g/L) and red cell distribution width (+0.40%). The effect of age on these mean deltas is negligible, while the levels of test results influence the difference between capillary and venous test results in most analytes. CONCLUSIONS: Our results improve guidance regarding the interpretation of capillary test results for children of all ages and in both physiological and pathological ranges.
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Índices de Eritrócitos , Flebotomia , Criança , Mineração de Dados , Contagem de Eritrócitos , Hematócrito , HumanosRESUMO
The quality and persistence of children's humoral immune response following SARS-CoV-2 infection remains largely unknown but will be crucial to guide pediatric SARS-CoV-2 vaccination programs. Here, we examine 548 children and 717 adults within 328 households with at least one member with a previous laboratory-confirmed SARS-CoV-2 infection. We assess serological response at 3-4 months and 11-12 months after infection using a bead-based multiplex immunoassay for 23 human coronavirus antigens including SARS-CoV-2 and its Variants of Concern (VOC) and endemic human coronaviruses (HCoVs), and additionally by three commercial SARS-CoV-2 antibody assays. Neutralization against wild type SARS-CoV-2 and the Delta VOC are analysed in a pseudotyped virus assay. Children, compared to adults, are five times more likely to be asymptomatic, and have higher specific antibody levels which persist longer (96.2% versus 82.9% still seropositive 11-12 months post infection). Of note, symptomatic and asymptomatic infections induce similar humoral responses in all age groups. SARS-CoV-2 infection occurs independent of HCoV serostatus. Neutralization responses of children and adults are similar, although neutralization is reduced for both against the Delta VOC. Overall, the long-term humoral immune response to SARS-CoV-2 infection in children is of longer duration than in adults even after asymptomatic infection.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Imunidade Humoral/imunologia , SARS-CoV-2/imunologia , Adolescente , Adulto , Antígenos Virais/imunologia , COVID-19/prevenção & controle , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Feminino , Humanos , Lactente , Masculino , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinação/métodosRESUMO
Reference intervals for laboratory test results have to be appropriate for the population in which they are used to be clinically useful. While sex and age are established partitioning criteria, patients' origin also influences laboratory test results, but is not commonly considered when creating or applying reference intervals. In the German population, stratification for ethnicity is rarely performed, and no ethnicity-specific hematology reference intervals have been reported yet. In this retrospective study, we investigated whether specific reference intervals are warranted for the numerically largest group of non-German descent, individuals originating from Turkey. To this end, we analyzed 1,314,754 test results from 167,294 patients from six German centers. Using a name-based algorithm, 1.9% of patients were identified as originating from Turkey, in line with census data and the algorithm's sensitivity. Reference intervals and their confidence intervals were calculated using an indirect data mining approach, and Turkish and non-Turkish reference limits overlapped completely or partially in nearly all analytes, regardless of age and sex, and only 5/144 (3.5%) subgroups' reference limits showed no overlap. We therefore conclude that the current practice of using common reference intervals is appropriate and allows correct clinical decision-making in patients originating from Turkey.
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Análise Química do Sangue , Emigrantes e Imigrantes , Etnicidade , Feminino , Alemanha/etnologia , Humanos , Masculino , Valores de Referência , Estudos Retrospectivos , Turquia/etnologiaRESUMO
OBJECTIVES: Assessment of children's laboratory test results requires consideration of the extensive changes that occur during physiological development and result in pronounced sex- and age-specific dynamics in many biochemical analytes. Pediatric reference intervals have to account for these dynamics, but ethical and practical challenges limit the availability of appropriate pediatric reference intervals that cover children from birth to adulthood. We have therefore initiated the multi-center data-driven PEDREF project (Next-Generation Pediatric Reference Intervals) to create pediatric reference intervals using data from laboratory information systems. METHODS: We analyzed laboratory test results from 638,683 patients (217,883-982,548 samples per analyte, a median of 603,745 test results per analyte, and 10,298,067 test results in total) performed during patient care in 13 German centers. Test results from children with repeat measurements were discarded, and we estimated the distribution of physiological test results using a validated statistical approach (kosmic). RESULTS: We report continuous pediatric reference intervals and percentile charts for alanine transaminase, aspartate transaminase, lactate dehydrogenase, alkaline phosphatase, γ-glutamyl-transferase, total protein, albumin, creatinine, urea, sodium, potassium, calcium, chloride, anorganic phosphate, and magnesium. Reference intervals are provided as tables and fractional polynomial functions (i.e., mathematical equations) that can be integrated into laboratory information systems. Additionally, Z-scores and percentiles enable the normalization of test results by age and sex to facilitate their interpretation across age groups. CONCLUSIONS: The provided reference intervals and percentile charts enable precise assessment of laboratory test results in children from birth to adulthood. Our findings highlight the pronounced dynamics in many biochemical analytes in neonates, which require particular consideration in reference intervals to support clinical decision making most effectively.
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Fosfatase Alcalina , gama-Glutamiltransferase , Adulto , Alanina Transaminase , Aspartato Aminotransferases , Criança , Humanos , Recém-Nascido , Valores de ReferênciaRESUMO
Highly sensitive and specific platforms for the detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies are becoming increasingly important for evaluating potential SARS-CoV-2 convalescent plasma donors, studying the spread of SARS-CoV-2 infections, and identifying individuals with seroconversion. This study provides a comparative validation of 4 anti-SARS-CoV-2 platforms. A unique feature of the study is the use of a representative cohort of convalescent patients with coronavirus disease 2019 and a mild to moderate disease course. All platforms showed significant correlations with a SARS-CoV-2 plaque reduction neutralization test, with highest sensitivities for the Euroimmun and the Roche platforms, suggesting their preferential use for screening persons at increased risk of SARS-CoV-2 infections.
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Teste Sorológico para COVID-19/normas , COVID-19/terapia , SARS-CoV-2/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Teste Sorológico para COVID-19/métodos , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Imunização Passiva/normas , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Doadores de Tecidos , Adulto Jovem , Soroterapia para COVID-19RESUMO
WHAT IS KNOWN AND OBJECTIVE: Delayed cerebral ischaemia is an important cause of morbidity and mortality after aneurysmal subarachnoid haemorrhage (aSAH). Nimodipine is the only drug approved by the FDA for improving outcome after aSAH. Clinically, however, there are no specific values of this drug in cerebrospinal fluid (CSF) during aSAH treatment that could be associated to outcome improvement. Furthermore, the neurotransmitter glutamate acts as a secondary marker for brain injury. The aim was to establish a method to measure nimodipine and glutamate concentrations simultaneously in CSF of patients after aSAH. METHODS: From June 2017 to June 2018, we prospectively collected clinical data of patients with aSAH admitted to our neurointensive care unit. All included patients received nimodipine orally (60 mg every 4 hours). Patients, who developed clinical vasospasm during their in-hospital stay, underwent intra-arterial application of nimodipine (IAN), followed by angiographic control. A method using high-performance liquid chromatography coupled with mass spectrometric analysis (LC-MS/MS) was established for quantification of both analytes in CSF. RESULTS AND DISCUSSION: In 15 (60%) of 25 patients, nimodipine and glutamate concentrations were measured. After IAN for treatment of vasospasms, CSF nimodipine concentrations were slightly higher than in patients who received nimodipine only orally (0.60 ± 0.27 ng/mL vs 0.48 ± 0.18 ng/mL). Patients developing vasospasm exhibited higher glutamate concentrations than patients without vasospasm (188.84 ng/mL vs136.07 ng/mL). WHAT IS NEW AND CONCLUSION: The developed method allowed the simultaneous quantification of nimodipine and glutamate in CSF. Furthermore, we demonstrated that IAN resulted in higher concentrations in CSF, when compared to oral application only.
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Ácido Glutâmico/líquido cefalorraquidiano , Nimodipina/administração & dosagem , Hemorragia Subaracnóidea/tratamento farmacológico , Espectrometria de Massas em Tandem/métodos , Adulto , Idoso , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/líquido cefalorraquidiano , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nimodipina/líquido cefalorraquidiano , Projetos Piloto , Estudos Prospectivos , Vasoespasmo Intracraniano/patologiaRESUMO
Background Interpreting hematology analytes in children is challenging due to the extensive changes in hematopoiesis that accompany physiological development and lead to pronounced sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, and limitations in current approaches to laboratory test result displays restrict their use when guiding clinical decisions. Methods We employed an improved data-driven approach to create percentile charts from laboratory data collected during patient care in 10 German centers (9,576,910 samples from 358,292 patients, 412,905-1,278,987 samples per analyte). We demonstrate visualization of hematology test results using percentile charts and z-scores (www.pedref.org/hematology) and assess the potential of percentiles and z-scores to support diagnosis of different hematological diseases. Results We created percentile charts for hemoglobin, hematocrit, red cell indices, red cell count, red cell distribution width, white cell count and platelet count in girls and boys from birth to 18 years of age. Comparison of pediatricians evaluating complex clinical scenarios using percentile charts versus conventional/tabular representations shows that percentile charts can enhance physician assessment in selected example cases. Age-specific percentiles and z-scores, compared with absolute test results, improve the identification of children with blood count abnormalities and the discrimination between different hematological diseases. Conclusions The provided reference intervals enable precise assessment of pediatric hematology test results. Representation of test results using percentiles and z-scores facilitates their interpretation and demonstrates the potential of digital approaches to improve clinical decision-making.
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Hematócrito/métodos , Hematologia/métodos , Hematologia/normas , Adolescente , Adulto , Criança , Pré-Escolar , Contagem de Eritrócitos , Índices de Eritrócitos , Feminino , Hematócrito/normas , Hemoglobinas/análise , Humanos , Lactente , Recém-Nascido , Contagem de Leucócitos , Masculino , Contagem de Plaquetas , Valores de Referência , Adulto JovemRESUMO
BACKGROUND: Interpretation of alkaline phosphatase activity in children is challenging due to extensive changes with growth and puberty leading to distinct sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics and seem reasonable for an analyte as closely linked to growth as alkaline phosphatase. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, resulting in limitations when clinical decisions are based on alkaline phosphatase activity. METHODS: We applied an indirect method to generate percentile charts for alkaline phosphatase activity using clinical laboratory data collected during the clinical care of patients. A total of 361,405 samples from 124,440 patients from six German tertiary care centers and one German laboratory service provider measured between January 2004 and June 2015 were analyzed. Measurement of alkaline phosphatase activity was performed on Roche Cobas analyzers using the IFCC's photometric method. RESULTS: We created percentile charts for alkaline phosphatase activity in girls and boys from birth to 18 years which can be used as reference intervals. Additionally, data tables of age- and sex-specific percentile values allow the incorporation of these results into laboratory information systems. CONCLUSIONS: The percentile charts provided enable the appropriate differential diagnosis of changes in alkaline phosphatase activity due to disease and changes due to physiological development. After local validation, integration of the provided percentile charts into result reporting facilitates precise assessment of alkaline phosphatase dynamics in pediatrics.
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Fosfatase Alcalina/análise , Pediatria , Adolescente , Fosfatase Alcalina/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Valores de ReferênciaRESUMO
Biofilms growing on copper and stainless steel substrata in natural drinking water were investigated. A modular pilot-scale distribution facility was installed at four waterworks using different raw waters and disinfection regimes. Three-month-old biofilms were analysed using molecular biology and microscopy methods. High total cell numbers, low counts of actively respiring cells and low numbers of cultivable bacteria indicated the high abundance of viable but not cultivable bacteria in the biofilms. The expression of the recA SOS responsive gene was detected and underlined the presence of transcriptionally active bacteria within the biofilms. This effect was most evident after UV disinfection, UV oxidation and UV disinfection with increased turbidity at waterworks compared to chemically treated and non-disinfected systems. Furthermore, live/dead staining techniques and environmental scanning electron microscopy imaging revealed the presence of living and intact bacteria in biofilms on copper substrata. Cluster analyses of DGGE profiles demonstrated differences in the composition of biofilms on copper and steel materials.
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Fenômenos Fisiológicos Bacterianos/efeitos da radiação , Biofilmes/efeitos da radiação , Cobre/química , Desinfecção/métodos , Microbiologia da Água , Purificação da Água/métodos , Bactérias/crescimento & desenvolvimento , Bactérias/efeitos da radiação , Biofilmes/crescimento & desenvolvimento , Água Potável , Aço Inoxidável/químicaRESUMO
Pseudothrombocytopenia remains a challenge in the haematological laboratory. The pre-analytical problem that platelets tend to easily aggregate in vitro, giving rise to lower platelet counts, has been known since ethylenediamine-tetra acetic acid EDTA and automated platelet counting procedures were introduced in the haematological laboratory. Different approaches to avoid the time and temperature dependent in vitro aggregation of platelets in the presence of EDTA were tested, but none of them proved optimal for routine purposes. Patients with unexpectedly low platelet counts or flagged for suspected aggregates, were selected and smears were examined for platelet aggregates. In these cases patients were asked to consent to the drawing of an additional sample of blood anti-coagulated with a magnesium additive. Magnesium was used in the beginning of the last century as anticoagulant for microscopic platelet counts. Using this approach, we documented 44 patients with pseudothrombocytopenia. In all cases, platelet counts were markedly higher in samples anti-coagulated with the magnesium containing anticoagulant when compared to EDTA-anticoagulated blood samples. We conclude that in patients with known or suspected pseudothrombocytopenia the magnesium-anticoagulant blood samples may be recommended for platelet counting.
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Anticoagulantes/farmacologia , Sulfato de Magnésio/farmacologia , Contagem de Plaquetas/métodos , Trombocitopenia/diagnóstico , Adolescente , Adulto , Idoso , Coleta de Amostras Sanguíneas/métodos , Erros de Diagnóstico/prevenção & controle , Ácido Edético/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/efeitos dos fármacos , Trombocitopenia/sangue , Adulto JovemRESUMO
Polymorphonuclear granulocytes (PMNs) have been attributed a primarily deleterious role in the pathogenesis of acute lung injury (ALI). However, evidence exists that PMNs might also act beneficially in certain types of ALI. In this regard, we investigated the role of activated neutrophils in the pathophysiology of lung contusion-induced ALI. We used the model of blunt chest trauma accompanied by PMN-depletion in male C3H/HeN mice. Animals received 25 µg/g body weight PMN-depleting antibody Gr-1 intravenously 48 h before trauma. Bronchoalveolar lavage (BAL) and lung tissue interleukin 6 (IL-6) were similarly elevated in PMN-depleted and control animals after trauma, whereas macrophage inflammatory protein 2 and monocyte chemoattractant protein 1 in BAL and lungs, IL-10 in BAL, and lung keratinocyte chemoattractant (KC) were even further increased in the absence of PMNs. Plasma IL-6 and KC were also increased in response to the insult and even further in the absence of PMNs. Chest trauma induced an enhanced release of IL-6, tumor necrosis factor α, macrophage inflammatory protein 2, monocyte chemoattractant protein 1, and IL-10 from isolated KU, which was blunted in the absence of PMNs. In the presence of PMNs, BAL protein was further increased at 30 h when compared with the 3-h time point, which was not the case in the absence of PMNs. Taken together, in response to lung trauma, activated neutrophils control inflammation including mediator release from distant immune cells but simultaneously mediate pulmonary tissue damage. Thus, keeping in mind potential inflammatory adverse effects, modulation of neutrophil activation or trafficking might be a reasonable therapeutic approach in chest trauma-induced lung injury.
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Granulócitos/citologia , Células de Kupffer/citologia , Lesão Pulmonar/metabolismo , Neutrófilos/citologia , Baço/citologia , Animais , Peso Corporal , Lavagem Broncoalveolar , Queratinócitos/citologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Peroxidase/metabolismo , Fatores de Tempo , Ferimentos não Penetrantes/metabolismoRESUMO
Surface expressed negatively charged sialoglycans contribute to the regulation of adhesive cellular interactions and are thus involved in the growth and differentiation of hematopoietic progenitor cells. In particular, the cell surface sialylation state may govern the liberation of CD34+ hematopoietic precursors from bone marrow stroma cells and extracellular matrix. In order to assess the overall surface sialylation of live human CD34+ hematopoietic precursor cells, we applied a previously described flow cytometric enzyme assay. Cells with and without sialidase pretreatment were incubated in the presence of fluorescent CMP-sialic acid and exogenous ST6GalI. Thus sialylation of surface-expressed lactosamine residues was analysed. We demonstrated that surface lactosamines of CD34+ precursors derived from bone marrow and peripheral blood are over 95% sialylated, predominantly in alpha2-6 linkage. These results are in accordance with flow cytometric analysis of surface lectin staining. Sialic acid specific lectins MAA and SNA were strongly bound whereas SBA, VVA, and PNA became reactive only after sialidase pretreatment. CD34+ leukemia cell lines TF1 and KG1a also showed a high degree of surface sialylation, whereas cell line KG1 expressed to the largest extent free lactosamines. In these cell lines, alpha2-6 and alpha2-3 sialylated residues were present in equal amounts. In a variation of the flow cytometric enzyme assay, live cells were incubated without exogenous STGal I to measure the activity of endogenous ecto-sialyltransferase. Ecto sialyltransferase activity was observed in all CD34+ cells which was able to resialylate major surface glycoproteins such as HLA Class I, CD45, CD43, and CD34. The ecto-sialyltransferase may serve to maintain or increase surface sialylation rapidly without de novo synthesis.
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Antígenos CD34/análise , Células-Tronco Hematopoéticas/química , Sialiltransferases/metabolismo , Antígenos CD34/sangue , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/sangue , Sialiltransferases/sangueRESUMO
The aim of this prospective study was to determine the local concentrations of inflammatory mediators in various tissue types frequently affected by trauma to estimate the role of prestored cytokine release by mechanical tissue trauma in the induction of a systemic inflammatory response syndrome. The degree of tissue damage, evaluated by its systemic release of inflammatory mediators, represents an important factor concerning the outcome of trauma patients. Clinical trials indicate that the kind of traumatized tissue influences the cytokine pattern measured in patients blood afterwards. However, the tissue-specific mediator composition underlying this systemic mediator release is rarely elucidated. Upon approval of the local IRB/EC, skin, subcutaneous fat, muscle, cancellous bone, and lung tissue were obtained during standard surgical procedures. The protein-based concentrations of Interleukin (IL)-6, IL-8, IL-10, and IL-12 were determined in tissue homogenates by enzyme-linked immunoabsorbant assay (ELISA; n = 60 samples). Albumin was measured to evaluate the degree of blood contamination of tissue samples. IL-6 and IL-8 were consistently detectable in more than 95% of the tissue specimens. Lung and cancellous bone presented by far the highest concentrations of these cytokines, whereas skin, subcutaneous fat, and muscle showed significantly lower levels. IL-10 was not detectable in 88%; IL-12 could not be measured in 63% of the samples. Cytokine concentrations did not correlate with the amount of albumin measured in tissue specimens. Due to their consistent presence at the tissue level, high systemic concentrations of IL-6 and IL-8 in patients blood, seen after pulmonary trauma, long bone fractures, or soft tissue injury, may be interpreted as an overspill of local trauma mediators. This indicates their relevance in post-traumatic monitoring. Furthermore, albumin is a suitable and necessary indicator to evaluate influences of possible blood contamination in tissue samples.
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Tecido Adiposo/química , Osso e Ossos/química , Citocinas/análise , Fraturas do Colo Femoral/metabolismo , Mediadores da Inflamação/análise , Neoplasias Pulmonares/metabolismo , Músculo Esquelético/química , Osteoartrite do Quadril/metabolismo , Pele/química , Ferimentos não Penetrantes , Idoso , Idoso de 80 Anos ou mais , Albuminas/análise , Fraturas do Colo Femoral/patologia , Fraturas do Colo Femoral/cirurgia , Humanos , Interleucina-10/análise , Interleucina-12/análise , Interleucina-6/análise , Interleucina-8/análise , Período Intraoperatório , Pulmão/química , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Pessoa de Meia-Idade , Especificidade de Órgãos , Osteoartrite do Quadril/patologia , Osteoartrite do Quadril/cirurgia , Estudos Prospectivos , Proteínas/análise , Síndrome de Resposta Inflamatória SistêmicaRESUMO
AIM: The aim of this study was to enhance the delivery and thus anti-tumoral efficiency of antisense bcl-2 oligonucleotides (ODN's). METHODS: Bcl-2 overexpressing DoHH2 lymphoma and HeLa-cells were transfected with ODN's using a polycationic liposome preparation. Specific hybridization of antisense ODN's was demonstrated by gel-shift assays and in vitro transcription/translation studies. Cellular uptake of oligonucleotides was evaluated by fluorescence microscopy. Inhibition of bcl-2 translation was demonstrated by quantitative RT-PCR and Western Blot. TUNEL assay, ANNEXIN V-binding and Apo-2.7 expression were performed to evaluate induction of apoptosis. RESULTS: Using polycationic liposomes, a ODN transfection rate of 95% in HeLa and 45% in DoHH2 cells were demonstrated by fluorescence microscopy. 24 hours after transfection quantitative RT-PCR detected a 56% decrease of bcl-2 mRNA in antisense and a 7% decrease in sense transfected DoHH2 cells (p < 0.05). In HeLa-cells, bcl-2 expression was almost completely inhibited 72 hours after antisense ODN transfection. Antisense treated cells also showed significant induction of apoptosis. CONCLUSIONS: Polycationic liposome-mediated transfection of bcl-2 antisense ODN's causes enhanced cellular uptake and efficient bcl-2 downregulation in bcl-2 overexpressing cell lines. This delivery strategy may explain why significant induction of apoptosis was achieved at low oligonucleotide concentrations (approximately 200 pmol/5 x 10(5) tumor cells).
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Apoptose/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Genes bcl-2/genética , Linfoma/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Anexina A5/metabolismo , Western Blotting , Primers do DNA/química , Regulação para Baixo , Ensaio de Desvio de Mobilidade Eletroforética , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Lipossomos , Linfoma/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The effect of long-term stagnation on copper corrosion by-product release and corrosion rates was studied in pipe-rigs according to the German standard DIN 50931, Part 1. The analysis of the water phase was supplemented by surface analysis of corrosion scales. Copper concentration during stagnation did not follow a solubility process. The characteristic curves obtained can be explained by subsequent copper release and copper refixation processes. Oxygen consumption can be described by the first-order kinetic rate law. The corrosion scales consisted of cuprite (Cu2O) and malachite (CuCO3 x Cu(OH)2). Malachite grew in well-defined crystals during stagnation, served as sink for dissolved copper and did not protect the pipe against corrosion attack. Copper concentrations measured after long-term stagnation (up to 122 h) correspond to the solubility of malachite in the testwater.