RESUMO
BACKGROUND: Systemic inflammation following curative surgery for colorectal cancer may be associated with increased risk of recurrence. [Correction added on 29 November 2019, after first online publication: text amended for accuracy.] This study investigated whether a clinically suspected infection, for which blood cultures were sent within 30 days after surgery for colorectal cancer, was associated with long-term oncological outcomes. METHODS: This register-based national cohort study included all Danish residents undergoing surgery with curative intent for colorectal cancer between January 2003 and December 2013. Patients who developed recurrence or died within 180 days after surgery were not included. Associations between blood cultures taken within 30 days after primary surgery and overall survival, disease-free survival and recurrence-free survival were analysed using Cox regression models adjusted for relevant clinical confounders, including demographic data, cancer stage, co-morbidity, blood transfusion, postoperative complications and adjuvant chemotherapy. RESULTS: The study included 21 349 patients, of whom 3390 (15·9 per cent) had blood cultures taken within 30 days after surgery. Median follow-up was 5·6 years. Patients who had blood cultures taken had an increased risk of all-cause mortality (hazard ratio (HR) 1·27, 95 per cent c.i. 1·20 to 1·35; P < 0·001), poorer disease-free survival (HR 1·22, 1·16 to 1·29; P < 0·001) and higher risk of recurrence (HR 1·15, 1·07 to 1·23; P < 0·001) than patients who did not have blood cultures taken. CONCLUSION: A clinically suspected infection requiring blood cultures within 30 days of surgery for colorectal cancer was associated with poorer oncological outcomes.
ANTECEDENTES: La inflamación sistémica en el cáncer colorrectal puede asociarse con un aumento del riesgo de recidiva. En este estudio se investigó si la sospecha clínica de infección, en la que se obtuvieron cultivos de sangre periférica durante los primeros 30 días de la cirugía por cáncer colorrectal, se asociaba con los resultados oncológicos a largo plazo. MÉTODOS: Se trata de un estudio de cohortes de un registro de una base de datos nacional, que incluyía todos los sujetos residentes en Dinamarca sometidos a cirugía por cáncer colorrectal con intención curativa desde enero de 2003 a diciembre de 2013. Los pacientes con recidiva o que fallecieron durante los primeros 180 días después de la cirugía fueron excluidos. Se estimaron las asociaciones entre los cultivos de sangre periférica efectuados en los primeros 30 días tras la cirugía primaria y la supervivencia global, supervivencia libre de enfermedad y supervivencia libre de recidiva mediante modelos de regresión de Cox, ajustados por variables clínicas confusoras relevantes (incluyendo datos demográficos, estadio del cáncer, comorbilidad, transfusión de sangre, complicaciones postoperatorias y quimioterapia adyuvante). RESULTADOS: El estudio incluyó 21.349 pacientes, de los cuales en 3.390 (16%) se habían obtenido cultivos de sangre periférica durante los primeros 30 días tras la cirugía. La mediana de seguimiento fue de 5,6 años. Los pacientes en los que se había obtenido cultivos de sangre periférica presentaron un riesgo aumentado de mortalidad por cualquier causa (cociente de riesgos instantáneos, hazard ratio, HR 1,27, i.c. del 95% 1,20-1,35; P < 0.0001), peor supervivencia libre de enfermedad (HR 1,22, i.c. del 95% 1,16-1,29; P < 0,0001) y mayor riesgo de recidiva (HR 1,15, i.c. del 95% 1,07-1,23; P < 0,0001) que los pacientes en los que no se habían obtenido cultivos. CONCLUSIÓN: La presencia de una infección sospechada clínicamente para la cual se requiere obtener cultivos de sangre periférica en los primeros 30 días tras cirugía por cancer colorrectal se asoció con peores resultados oncológicos.
Assuntos
Colectomia , Neoplasias Colorretais/cirurgia , Complicações Pós-Operatórias/sangue , Sistema de Registros , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Hemocultura , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Dinamarca/epidemiologia , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/mortalidade , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida/tendências , Fatores de TempoRESUMO
Early changes in biomarker levels probably occur before bloodstream infection (BSI) is diagnosed. However, this issue has not been fully addressed. We aimed at evaluating the kinetics of C-reactive protein (CRP) and plasma albumin (PA) in the 30 days before community-acquired (CA) BSI diagnosis. From a population-based BSI database we identified 658 patients with at least one measurement of CRP or PA from day -30 (D-30) through day -1 (D-1) before the day of CA-BSI (D0) and a measurement of the same biomarker at D0 or D1. Amongst these, 502 had both CRP and PA measurements which fitted these criteria. CRP and PA concentrations began to change inversely some days before CA-BSI diagnosis, CRP increasing by day -3.1 and PA decreasing by day -1.3. From D-30 to D-4, CRP kinetics (expressed as slopes - rate of concentration change per day) was -1.5 mg/l/day. From D-3 to D1, the CRP slope increased to 36.3 mg/l/day. For albumin, the slope between D-30 to D-2 was 0.1 g/l/day and changed to -1.8 g/l/day between D-1 and D1. We showed that biomarker levels begin to change some days before the CA-BSI diagnosis, CRP 3.1 days and PA 1.3 days before.
Assuntos
Bacteriemia/patologia , Biomarcadores/sangue , Proteína C-Reativa/análise , Infecções Comunitárias Adquiridas/patologia , Período de Incubação de Doenças Infecciosas , Albumina Sérica/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Dinamarca , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Community-acquired bacteraemia patients (n = 2472), Denmark, 2000-2008. Albumin, C-reactive protein (CRP) and haemoglobin (Hb) measured 2000-2010. We assessed daily mean levels of albumin, CRP and Hb from 30 days before to 30 days after bacteraemia and correlations between albumin vs. CRP and albumin vs. Hb. In linear regression models, we evaluated the contribution of CRP, Hb, chronic and acute variables to the albumin level variations. The mean albumin level (33.6 g/l) was steady before day 1, declined to 29.3 g/l on day 1 with little increase afterward. The mean CRP increased from day -5, peaked on day 1 and declined thereafter. The mean Hb level was fairly constant during days -30/30. Albumin was inversely (R range, - 0.18/-0.47, P < 10-4) correlated with the CRP level and positively (R = 0.17-0.46, P < 10-4) correlated with the HB level. In most models, CRP was the first variable that contributed to the albumin variations, 34-70% of the full model. The sudden decrease of albumin levels, without sudden fluctuations of CRP or Hb, indicated that hypoalbuminaemia was a marker of trans-capillary leakage.
Assuntos
Bacteriemia/complicações , Proteína C-Reativa/análise , Hipoalbuminemia/etiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/microbiologia , Biomarcadores/sangue , Infecções Comunitárias Adquiridas/complicações , Infecções Comunitárias Adquiridas/microbiologia , Dinamarca , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Vigilância da População , Adulto JovemRESUMO
This study compared Neo-Sensitabs with Oxoid paper disks using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion antimicrobial susceptibility test on Mueller-Hinton agar. The EUCAST-recommended quality control strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) (Part I) and clinical isolates (Part II) were investigated. In Part I of the study, 27 combinations of antimicrobial agents were tested on four quality control strains repeatedly up to 60 times and zone diameters of tablets and disks were compared. In Part II of the study, 351 clinical isolates were included to cover a broad range of species, as well as resistance mechanisms. In Part I, four major deviations (>1 mm outside quality control ranges) were observed with Neo-Sensitabs. In one case with P. aeruginosa ATCC 27853 (meropenem), there was a corresponding major deviation (2 mm) with the Oxoid disk. The three remaining major deviations with Neo-Sensitabs were observed with meropenem (2 mm) in E. coli ATCC 25922 and with ciprofloxacin (2 mm) and gentamicin (3 mm) in P. aeruginosa ATCC 27853. For Oxoid disks, there were only minor deviations (=1 mm outside quality control ranges) in these three cases. In Part II, there were six discrepancies, susceptible versus resistant, in 3,533 comparisons between the two methods with the clinical isolates. The Rosco Neo-Sensitabs appear to be a possible alternative to Oxoid paper disks for EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.
Assuntos
Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/instrumentação , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/métodos , Ágar , Infecções Bacterianas/microbiologia , Meios de Cultura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão/normas , HumanosRESUMO
We conducted a hospital-based cohort study among adult patients with first-time Streptococcus pneumoniae bacteremia (SPB) from 2000 through 2008. Patients were identified in a population-based bacteremia database and followed up for mortality through the Danish Civil Registration System (CRS). The aim of the study was to determine the focal diagnosis of SPB, the severity of sepsis at presentation, demographics and comorbidity characteristics of the patients, and to determine the 30-day mortality rate and factors related to mortality. We identified 481 patients, of which 238 were males. The mean age of the patients was 65 years. The focal diagnosis of the SPB was pneumonia in 381 (79 %) patients, followed in frequency by meningitis in 33 (7 %) patients. Of the 481 patients, 390 (81 %) had community-acquired SPB. Of these, 23 (6 %) did not have sepsis, 132 (34 %) had sepsis, 224 (57 %) had severe sepsis, and 11 (3 %) were in septic shock. Overall, the 30-day mortality was 16 %. Mortality increased with the severity of sepsis. There was no association between the focal diagnosis of SPB or the number of diagnoses and mortality. Nosocomial infection, male sex, increasing age, and increasing comorbidity were all associated with an increased 30-day mortality rate.
Assuntos
Bacteriemia/diagnóstico , Bacteriemia/mortalidade , Sistema de Registros/normas , Sepse/microbiologia , Streptococcus pneumoniae/patogenicidade , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/microbiologia , Estudos de Coortes , Infecções Comunitárias Adquiridas/microbiologia , Infecções Comunitárias Adquiridas/mortalidade , Comorbidade , Infecção Hospitalar/microbiologia , Infecção Hospitalar/mortalidade , Dinamarca/epidemiologia , Feminino , Hospitais Universitários , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Pneumocócica/diagnóstico , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/mortalidade , Modelos de Riscos Proporcionais , Fatores de Risco , Índice de Gravidade de Doença , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/isolamento & purificação , Fatores de Tempo , Adulto JovemRESUMO
In Denmark recurrent epidemics of Mycoplasma pneumoniae infections have been described since the 1950s at intervals of approximately four to six years. The latest epidemic occurred in 2004/05 followed by two years of high incidence and more than three years of low incidence. Due to a recent increase in diagnosed cases since late summer 2010, we conducted a survey of positive M. pneumoniae PCR tests performed by clinical microbiology departments in Denmark, which indicated that a new epidemic may be underway.
Assuntos
Epidemias/estatística & dados numéricos , Mycoplasma pneumoniae/isolamento & purificação , Pneumonia por Mycoplasma/epidemiologia , Vigilância da População , Coleta de Dados , Dinamarca/epidemiologia , Humanos , Incidência , Laboratórios , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/imunologia , Pneumonia por Mycoplasma/diagnóstico , Pneumonia por Mycoplasma/microbiologia , Reação em Cadeia da PolimeraseRESUMO
Acquisition of Legionnaires' disease is a serious complication of hospitalization. Rapid determination of whether or not the infection is caused by strains of Legionella pneumophila in the hospital environment is crucial to avoid further cases. This study investigated the use of whole-genome sequencing to identify the source of infection in hospital-acquired Legionnaires' disease. Phylogenetic analyses showed close relatedness between one patient isolate and a strain found in hospital water, confirming suspicion of nosocomial infection. It was found that whole-genome sequencing can be a useful tool in the investigation of hospital-acquired Legionnaires' disease.
Assuntos
Infecção Hospitalar/microbiologia , Microbiologia Ambiental , Legionella pneumophila/classificação , Doença dos Legionários/microbiologia , Epidemiologia Molecular/métodos , Tipagem Molecular/métodos , Sequenciamento Completo do Genoma/métodos , Análise por Conglomerados , Humanos , Legionella pneumophila/genética , Legionella pneumophila/isolamento & purificação , Filogenia , Homologia de SequênciaRESUMO
BACKGROUND: A prerequisite for a satisfying functional result in the treatment of comminuted fractures of the proximal humerus with hemiarthroplasty is anatomical reduction, fixation and healing of the tuberculi around the prosthetic neck in order to restore normal function of the rotator cuff. PURPOSE: This was a retrospective study to examine the outcome after hemiarthroplasty using a prosthetic stem designed to optimise re-attachment and healing of the tuberculi (Aequalis; Tornier and Global Fx, DePuy). A special emphasis was on the effect on outcome a comminuted greater tubercle might have. MATERIALS AND METHODS: At follow-up, clinical results were evaluated using the Constant score and WOOS index. All patients had radiographs taken of the injured shoulder. Quality of tubercle healing and prosthetic height were estimated; acromiohumeral distance was registered as well as greater tubercle comminution and resorption. RESULTS: Thirty-four patients with 35 hemiarthroplasties were included. Mean age was 71 years (range 47-88) at the time of injury. At follow-up (mean 38 months, range 23-67), the mean Constant score was 44 points (range 18-87). The mean WOOS index was 58 (range 15-96). A comminuted tubercle was associated with tubercle resorption and superior migration of the arthroplasty. Also, there was a correlation for the functional Constant score, but for the WOOS index, there was none. CONCLUSION: Like several other studies, we generally saw a group of patients with limited pain but poor range of movement in the shoulder. Our hypothesis was that comminution of the greater tubercle would correlate with both rotator cuff arthropathic radiographical features and more detrimental functional scores than average. Thus, a subtype of fracture could be identified at the time of injury and perhaps be allocated to a different treatment than hemiarthroplasty. Due to a limited number of patients in this study, we are unable to make any strong statistically supported conclusions regarding this hypothesis. LEVEL OF EVIDENCE: Level 4 evidence.
Assuntos
Hemiartroplastia , Fraturas do Úmero/cirurgia , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Úmero/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Articulação do Ombro/cirurgiaRESUMO
An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.
Assuntos
Acil-CoA Desidrogenases/genética , Mutação , Acil-CoA Desidrogenase , Animais , Northern Blotting , Western Blotting , Linhagem Celular Transformada , Clonagem Molecular , DNA , Células Eucarióticas , Vetores Genéticos , Herpesvirus Humano 4/genética , Homozigoto , Humanos , Regiões Promotoras Genéticas , RNA/genética , Vírus 40 dos Símios/genética , TransfecçãoRESUMO
The influence of co-overexpression of the bacterial chaperonins GroEL and GroES on solubility, tetramer formation and enzyme activity of three variants of heterologously-expressed human medium-chain acyl-CoA dehydrogenase (MCAD) was analysed in order to investigate the molecular mechanism underlying MCAD deficiency caused by the prevalent K304E mutation. Depending on which of the three amino acids--lysine (wild-type), glutamic acid (K304E) or glutamine (K304Q) are present at position 304 of the mature polypeptide, three different patterns were observed in our assay system: (i) solubility, tetramer formation and yield of enzyme activity of wild-type MCAD is largely independent of GroESL co-overexpression; (ii) the larger part of the K304Q mutant is insoluble without and solubility is enhanced with GroESL co-overexpression; solubility correlates with the amount of tetramer detected and the enzyme activity measured as observed for the wild-type protein. (iii) Solubility of the K304E mutant is in a similar fashion GroESL responsive as the K304Q mutant, but the amount of tetramer observed and the enzyme activity measured do not correlate with the amount of soluble K304E MCAD protein detected in Western blotting. In a first attempt to estimate the specific activity, we show that tetrameric K304E and K304Q mutant MCAD display a specific activity in the range of the wild-type enzyme. Taken together, our results strongly suggest, that the K304E mutation primarily impairs the rate of folding and subunit assembly. Based on the data presented, we propose that lysine-304 is important for the folding pathway and that an exchange of this amino acid both to glutamine or glutamic acid leads to an increased tendency to misfold/aggregate. Furthermore, exchange of lysine-304 with an amino acid with negative charge at position 304 (glutamic acid) but not with a neutral charge (glutamine) negatively affects conversion to active tetramers. A possible explanation for this latter effect--charge repulsion upon subunit docking--is discussed.
Assuntos
Acil-CoA Desidrogenases/genética , Proteínas de Bactérias/metabolismo , Chaperoninas/metabolismo , Escherichia coli/genética , Proteínas/metabolismo , Acil-CoA Desidrogenase , Acil-CoA Desidrogenases/química , Acil-CoA Desidrogenases/metabolismo , Sequência de Bases , Expressão Gênica , Glutamatos , Ácido Glutâmico , Glutamina , Humanos , Lisina , Dados de Sequência Molecular , Mutação , Plasmídeos , Dobramento de ProteínaRESUMO
Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.
Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos , Atrofia Girata/enzimologia , Queratinócitos/metabolismo , Vírus da Leucemia do Macaco Gibão/genética , Ornitina-Oxo-Ácido Transaminase/genética , Células 3T3 , Animais , Terapia Genética , Atrofia Girata/terapia , Humanos , Camundongos , Ornitina-Oxo-Ácido Transaminase/deficiência , Ornitina-Oxo-Ácido Transaminase/metabolismoRESUMO
To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.
Assuntos
Transplante de Células , Células Epidérmicas , Epiderme/transplante , Técnicas de Transferência de Genes , Tolerância Imunológica , Animais , Células Cultivadas , Feminino , Genes Reporter , Humanos , Queratinócitos/fisiologia , Queratinócitos/transplante , Camundongos , Camundongos Nus , Transfecção , beta-Galactosidase/genéticaRESUMO
The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by postnatal onset of polyuria and a deficient neurosecretion of the antidiuretic hormone, arginine vasopressin (AVP). Since 1991, adFNDI has been linked to 31 different mutations of the gene that codes for the vasopressin-neurophysin II (AVP-NPII) precursor. The aims of the present study were to relate the clinical phenotype to the specific genotype and to the molecular genetic effects of the most frequently reported adFNDI mutation located at the cleavage site of the signal peptide of AVP-NPII [Ala(-1)Thr]. Genetic analysis and clinical studies of AVP secretion, urinary AVP, and urine output were performed in 16 affected and 16 unaffected family members and 11 spouses of a Danish adFNDI kindred carrying the Ala(-1)Thr mutation. Mutant complementary DNA carrying the same mutation was expressed in a neurogenic cell line (Neuro2A), and the cellular effects were studied by Western blotting, immunocytochemistry, and AVP measurements. The clinical studies showed a severe progressive deficiency of plasma and urinary AVP that manifested during childhood. The expression studies demonstrated that the Ala(- 1)Thr mutant cells produced 8-fold less AVP than wild-type cells and accumulated excessive amounts of 23-kDa NPII protein corresponding to uncleaved prepro-AVP-NPII. Furthermore, a substantial portion of the intracellular AVP-NPII precursor appeared to be colocalized with an endoplasmic reticulum antigen (Grp78). These results provide independent confirmation that this Ala(-1)Thr mutation produces adFNDI by directing the production of a mutant preprohormone that accumulates in the endoplasmic reticulum, because it cannot be cleaved from the signal peptide and transported to neurosecretory vesicles for further processing and secretion.
Assuntos
Arginina Vasopressina/genética , Diabetes Insípido/genética , Mutação de Sentido Incorreto , Neurofisinas/genética , Precursores de Proteínas/genética , Vasopressinas/genética , Adolescente , Adulto , Idoso , Arginina Vasopressina/metabolismo , Criança , Pré-Escolar , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neurofisinas/análise , Neurofisinas/metabolismo , Precursores de Proteínas/metabolismo , Vasopressinas/metabolismoRESUMO
The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of an LDL receptor mutation (W556S) found in FH patients and characterised as transport defective. In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-localisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum. Wild type (WT) and W556S LDL receptor GFP fusion proteins were expressed in mouse liver by means of hydrodynamic delivery of naked DNA. Two days after injection liver samples were analysed for GFP fluorescence. The WT LDL receptor GFP protein was located on the cell surface whereas the W556S LDL receptor GFP protein was retained in intracellular compartments. Thus, the GFP-tagged LDL receptor protein allows both detailed time lapse analysis and evaluations in animals for the physiological modelling of mutations. This method should be generally applicable in functional testing of gene products for aberrant processing.
Assuntos
Receptores de LDL/fisiologia , Animais , Transporte Biológico , Linhagem Celular , Endocitose , Genótipo , Proteínas de Fluorescência Verde , Humanos , Hiperlipoproteinemia Tipo II/genética , Lipoproteínas LDL/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Microscopia Confocal , Mutação , Receptores de LDL/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.
Assuntos
Hiperlipoproteinemia Tipo II/genética , Mutação Puntual , Receptores de LDL/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Sequência Conservada , Dinamarca , Éxons , Citometria de Fluxo , Imunofluorescência , Humanos , Microscopia Confocal , Sequências Repetitivas de Ácido Nucleico , Serina , Transfecção , TriptofanoRESUMO
The susceptibility testing methods used in Denmark were evaluated with respect to their ability to detect cephalosporin resistance with cefuroxime as indicator, especially resistance caused by extended-spectrum beta-lactamases (ESBLs). Two methods for determination of minimal inhibitory concentration (MIC), three agar diffusion methods, a disc approximation test and the ESBL-Etest were used against a panel of strains producing well-known beta-lactamases. The tablet diffusion test (Rosco Neo Sensitabs) as the most used in Denmark had the lowest detection rate of cefuroxime resistance among ESBL-producing strains. The prediffusion method, which is only used at one laboratory, was the most reliable method for such detection. The MIC methods were in good agreement, but the detection rate for resistance due to ESBLs was low and depended on the antibiotics used. The disc approximation test and the ESBL-Etest both resulted in an acceptable ESBL detection rate. The latter tests discriminated between isolates producing the frequent chromosome-mediated and the in Denmark probably very rare ESBL-mediated cephalosporin resistance. For the evaluation of susceptibility tests such strains require special attention.
Assuntos
Resistência às Cefalosporinas , beta-Lactamases/análise , Alcaligenes/efeitos dos fármacos , Alcaligenes/crescimento & desenvolvimento , Cefalosporinas/farmacologia , Dinamarca , Difusão , Enterobacter/efeitos dos fármacos , Enterobacter/crescimento & desenvolvimento , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Reprodutibilidade dos Testes , ComprimidosRESUMO
The apolipoprotein E (apoE) gene has been identified as a susceptibility gene for Alzheimer's disease, although the mechanism whereby apoE exerts this effect has not been determined. The aim of this study was to examine the intracellular fate of apoE and we demonstrate that in mammalian cells-transiently expressing tau and the low density lipoprotein receptor, apoE is taken up from cerebrospinal fluid (CSF) and apoE3, but not apoE4, reaches the cytoplasm. This isoform difference in the intracellular distribution of apoE is dependent upon tau expression and, although tau phosphorylation was not affected by apoE, these results suggest that apoE interactions with the cytoskeleton might be important in the molecular pathogenesis of Alzheimer's disease.
Assuntos
Doença de Alzheimer/genética , Apolipoproteínas E/genética , Proteínas tau/fisiologia , Alelos , Animais , Apolipoproteínas E/metabolismo , Células COS , Predisposição Genética para Doença , Humanos , FosforilaçãoRESUMO
Fusarium spp. cause infections only rarely in immunologically competent hosts, but disseminated infection may occur in severely immunocompromised patients. Symptoms of disseminated infection are persistent fever, despite broad-spectrum antibacterial and antifungal treatment, associated with skin lesions, most commonly on the extremities, in 60-80% of patients. A mortality rate of 50-75% has been reported for patients with disseminated fusariosis. Despite treatment failures, amphotericin B remains the preferred drug, in part because of lack of alternatives. Voriconazole is a promising new agent, but more clinical experience is required.
Assuntos
Fungemia/diagnóstico , Fusarium/isolamento & purificação , Doenças Hematológicas/complicações , Hospedeiro Imunocomprometido , Idoso , Dinamarca , Feminino , Fungemia/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Micoses/diagnóstico , Micoses/microbiologiaRESUMO
Alzheimer's disease with late onset is associated with inheritance of isoform 4 of the polymorphic apolipoprotein E (ApoE). We describe an experimental model where intracellular interaction between ApoE isoforms and cellular proteins may be studied. Internalization and intracellular fate of ApoE from fresh normal cerebrospinal fluid is followed in COS cells (an SV40 transformed Simian kidney cell line) overexpressing the low density lipoprotein receptor. Chase and chloroquine experiments strongly suggest that internalized ApoE travels through the endosomal-lysosomal pathway after dissociation from the receptor in early endosome.
Assuntos
Apolipoproteínas E/metabolismo , Cloroquina/farmacologia , Endocitose/efeitos dos fármacos , Receptores de LDL/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular Transformada , Células Cultivadas , Chlorocebus aethiops , Expressão Gênica , Lisossomos/metabolismo , Receptores de LDL/genética , TransfecçãoRESUMO
In 1997, 80 and 89% of Staphylococcus aureus isolated from university hospitals in Odense and Hvidovre respectively were resistant to penicillin and 32.0 and 41% of Escherichia coli were resistant to ampicillin. There were low incidences of methicillin resistance in S. aureus (<1%), penicillin resistance in Streptococcus pneumoniae (3%),and gentamicin in E. coli (2%). These figures might reflect the low use of antibiotics in Denmark.