RESUMEN
BACKGROUND: Elevation of CXCL13, a key regulator of B-cell recruitment in cerebrospinal fluid (CSF) is implicated in multiple sclerosis (MS). OBJECTIVE: to evaluate if measurement of CXCL13 using a highly sensitive assay is of value in acute optic neuritis (ON) patients for the prediction of later MS. METHOD: CXCL13 was measured by Simoa in two independent treatment-naïve ON cohorts, a training cohort (TC, n = 33) originating from a population-based cohort, a validation cohort (VC, n = 30) consecutively collected following principles for population studies. Prospectively, 14/33 TC and 12/30 VC patients progressed to MS (MS-ON) while 19/33 TC and 18/30 VC patients, remained as isolated ON (ION). RESULTS: CXCL13 was detectable in all samples and were higher in ON compared with healthy controls (HC) (p = 0.012). In the TC, CSF levels in MS-ON were higher compared with ION patients and HC (p = 0.0001 and p<0.0001). In the VC, we confirmed the increase of CXCL13 in MS-ON compared to ION (p = 0.0091). Logistic regression analysis revealed an area under receiver operating characteristic curve of 0.83 [95% C.I: 0.73-0.93]. CONCLUSIONS: The highly sensitive CXCL13 Simoa assay demonstrated ability to identify ON patients and separate MS-ON from ION, and predictive diagnostic values indicates a promising potential of this assay.
Asunto(s)
Esclerosis Múltiple , Neuritis Óptica , Biomarcadores , Quimiocina CXCL13 , Estudios de Cohortes , Humanos , Esclerosis Múltiple/diagnóstico , Neuritis Óptica/diagnóstico , Curva ROCRESUMEN
BACKGROUND: Vascular endothelial growth factor (VEGF) is implicated in airway remodelling and asthma development. We studied VEGFA gene variants and plasma levels and the development of lung function, bronchial hyperresponsiveness and asthma in childhood. METHODS: We analysed 13 SNPs in the VEGFA gene in 411 children from the COPSAC2000 high-risk birth cohort. Asthma was diagnosed prospectively, and lung function measurements were obtained at birth and 6 years of age. Plasma VEGF levels were measured at 18 months of age. We used a Bonferroni adjusted significance level. Findings were replicated in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) birth cohort at age 8. RESULTS: At age six, three SNPs from the same linkage block were associated with FEV1 (rs699947, P = 1.31E-05), independent of asthma, and there were suggestive associations between FEV1/FVC ratio and rs833052 and maximal mid-expiratory flow and rs6900017. Replication in the PIAMA cohort showed borderline association between FEV1 and rs699947 and significant meta-analysis result. SNPs upstream and nearby rs699947 were nominally associated with VEGF plasma levels. VEGF levels were not associated with asthmatic symptoms or lung function measures. CONCLUSIONS AND CLINICAL RELEVANCE: VEGF gene variants are associated with lung function at school age, but not at birth, suggesting a role of VEGF in post-natal lung function development.
Asunto(s)
Asma/genética , Asma/fisiopatología , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/fisiopatología , Variación Genética , Factor A de Crecimiento Endotelial Vascular/genética , Factores de Edad , Preescolar , Femenino , Humanos , Recién Nacido , Desequilibrio de Ligamiento , Masculino , Metaanálisis como Asunto , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Pruebas de Función Respiratoria , Factores de RiesgoRESUMEN
Tissue microarrays are a method of relocating tissue from conventional histologic paraffin blocks in a manner that tissue from multiple patients or blocks can be seen on the same slide. This is done by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block. This technique allows maximization of tissue resources by analysis of small core biopsies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed using cases from pathology tissue block archives, and a 20-year survival analysis can be done on a cohort of 600 or more patients using only a few microliters of antibody in a single experiment. Furthermore, this cohort can be analyzed thousands of times with different reagents as a result of judicious sectioning of the array block. This review describes this process and discusses the issues of representative sampling in heterogeneous lesions, the issue of antigen preservation, and some technical strategies and methods of array construction. In summary, this technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential for allowing validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.
Asunto(s)
Técnicas Histológicas/métodos , Neoplasias/genética , Neoplasias/patología , Patología/métodos , Técnicas Histológicas/instrumentación , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Patología/instrumentaciónRESUMEN
Tissue microarrays are a method of harvesting small disks of tissue from a range of standard histologic sections and placing them in an array on a recipient paraffin block such that hundreds of cases can be analyzed simultaneously. This technique allows maximization of tissue resources by analysis of small-core biop sies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed with cases from pathology tissue block archives, such that a 20-year survival analysis can be performed on a cohort of 600 or more patients by use of only a few microliters of antibody in a single experiment. The reflex criticism of this technique is that the tissue analyzed is not representative, especially in antigens with heterogeneous staining patterns. This review addresses this issue, as well as the issue of antigen preservation or durability, which validates construction of arrays from archives. Strategies and methods of construction and analysis of the arrays are discussed, as well as some other unusual array applications. This technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential to allow validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Técnicas de Preparación Histocitológica/métodos , Neoplasias/patología , Lesiones Precancerosas/patología , Análisis de Secuencia/métodos , Antígenos/análisis , Células Sanguíneas/patología , Técnica del Anticuerpo Fluorescente/métodos , Humanos , Técnicas para Inmunoenzimas , Neoplasias/químicaRESUMEN
A family of protein kinases regulates translation in response to different cellular stresses by phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF-2alpha). In yeast, an eIF-2alpha kinase, GCN2, functions in translational control in response to amino acid starvation. It is thought that uncharged tRNA that accumulates during amino acid limitation binds to sequences in GCN2 homologous to histidyl-tRNA synthetase (HisRS) enzymes, leading to enhanced kinase catalytic activity. Given that starvation for amino acids also stimulates phosphorylation of eIF-2alpha in mammalian cells, we searched for and identified a GCN2 homologue in mice. We cloned three different cDNAs encoding mouse GCN2 isoforms, derived from a single gene, that vary in their amino-terminal sequences. Like their yeast counterpart, the mouse GCN2 isoforms contain HisRS-related sequences juxtaposed to the kinase catalytic domain. While GCN2 mRNA was found in all mouse tissues examined, the isoforms appear to be differentially expressed. Mouse GCN2 expressed in yeast was found to inhibit growth by hyperphosphorylation of eIF-2alpha, requiring both the kinase catalytic domain and the HisRS-related sequences. Additionally, lysates prepared from yeast expressing mGCN2 were found to phosphorylate recombinant eIF-2alpha substrate. Mouse GCN2 activity in both the in vivo and in vitro assays required the presence of serine-51, the known regulatory phosphorylation site in eIF-2alpha. Together, our studies identify a new mammalian eIF-2alpha kinase, GCN2, that can mediate translational control.
Asunto(s)
Factor 2 Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Histidina-ARNt Ligasa/genética , Ratones , Datos de Secuencia Molecular , Mutación , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
OBJECTIVE: To examine outcomes and predictors of smoking cessation among elderly patients treated for nicotine dependence. DESIGN: Retrospective analysis of patients aged 65-82 who received a nicotine dependence consultation at the Mayo Medical Center between 1 April 1988 and 30 May 1992. Patients were contacted by telephone by a trained interviewer six months after the consultation and were sent a follow-up survey in August 1993. SETTING: Mayo Medical Center, Rochester, Minnesota, United States. SUBJECTS: A total of 613 patients (310 men, 303 women) with a mean age of 69.0 (SD 3.5) years were seen during the study period. MAIN OUTCOME MEASURES: Point prevalence self-reported smoking status. Patients were considered abstinent if they self-reported not smoking (not even a puff) during the seven days before contact. RESULTS: At six-month follow up, 24.8% of the 613 patients reported abstinence from smoking. On multivariate analysis, smoking abstinence was more likely if patients were hospitalised at the time of the consultation, married to a non-smoking spouse, very motivated to stop smoking, and reported their longest time of previous abstinence to be less than a day or more than a month. The response rate to the mailed follow-up survey was 69.9% (429 of 613). The mean duration of follow up was 40.0 +/- 13.2 months following the consultation. Of the 429 patients, 103 (24.0%) reported abstinence from smoking and 326 (76.0%) were smoking at six-month follow up. Patients who reported abstinence at six months had a higher cessation rate at the last follow up (76.0%) compared with patients who were smoking at six-month follow up (33.0%, P < 0.001). For patients who were not smoking at six months, no factors were found to significantly predict abstinence at last follow up. For patients who were smoking at six months, factors associated with smoking cessation at last follow up were: more than a year as the longest time off cigarettes before the consultation; counsellor rating of less severe nicotine dependence; and older age at first regular smoking. CONCLUSIONS: Several predictors of smoking cessation were identified in this study which may be useful for tailoring smoking interventions for the elderly.
Asunto(s)
Anciano , Nicotina , Cese del Hábito de Fumar , Trastornos Relacionados con Sustancias/rehabilitación , Atención Ambulatoria , Femenino , Hospitalización , Humanos , Masculino , Pronóstico , Estudios Retrospectivos , Índice de Severidad de la EnfermedadRESUMEN
Cholestyramine, an ion exchange resin shown to bind bacterial toxins, was utilized to treat rabbits with antibiotic induced enterotoxaemia. Three groups of 6 rabbits were administered 30 mg/kg clindamycin phosphate intravenously on day 1. One group was untreated; 2 groups were treated daily by gavage with 2 g cholestyramine in 20 ml water until day 21, starting on either day 1 or 3. Daily body weights, faecal output, faecal occult blood, food and water consumption, and body temperatures were determined. Four of 6 rabbits in the untreated group either died or were moribund and euthanased. There were no deaths in either treatment groups. Dramatic decreases in food consumption (86%), water consumption (62%), and faecal output (89%) were noted within 3 days after clindamycin administration in all groups. These parameters remained depressed throughout the study. There was no clear trend in body weight changes, body temperature, or faecal occult blood test results. Cholestyramine was effective in eliminating mortality associated with the intravenous administration of clindamycin and is recommended to prevent the development of enterotoxaemia when pyrogen testing or administering antibiotics known to induce the syndrome in rabbits.
Asunto(s)
Resina de Colestiramina/uso terapéutico , Clindamicina , Enterotoxemia/prevención & control , Conejos/microbiología , Animales , Peso Corporal/efectos de los fármacos , Enfermedades del Ciego/patología , Enfermedades del Ciego/veterinaria , Conducta de Ingestión de Líquido/efectos de los fármacos , Enterotoxemia/inducido químicamente , Enterotoxemia/mortalidad , Heces , Conducta Alimentaria/efectos de los fármacos , MasculinoRESUMEN
Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.
Asunto(s)
Cromosomas Humanos Par 20 , Anemia de Fanconi/genética , Ligamiento Genético , Mapeo Cromosómico , Femenino , Marcadores Genéticos/genética , Humanos , Escala de Lod , Masculino , LinajeRESUMEN
Seven monoclonal antibodies have been used for the serotyping of one hundred Neisseria gonorrhoeae wild strains, randomly selected from nine U.S. cities, and seven serotype reference strains by the co-agglutination method. As determined by gel-immunoradioassay, the monoclonal antibodies recognized the protein I trimer of a single or a limited subset of serotype reference strains. All but three strains were typable by one or two of the antibodies. The most common serotypes were 1.3 (26%), 1 (20%), 5 (17%), 5.7 (11%) and 9 (10%). To correlate typing results with ability for killing of these antibodies, susceptibility of typed and non-typed strains to killing was studied. Susceptibility was significantly associated with typing by the serotype 7 (p = 0.011) and serotype 9 (p = 0.033) specific monoclonal antibodies. Reaction of antibodies recognizing epitopes on the protein IB molecule with a given strain predicted in an average of 43% of strains (49% of strains of serotype 5, 62% of serotype 7, 29% of serotype 8, and 33% of serotype 9) its susceptibility to killing by the typing antibodies. In contrast, only 15% of the strains (15% of strains of serotype 1 and 15% of serotype 3) were killed by their typing antibodies, recognizing epitopes on the protein IA molecule. These monoclonal antibodies might prove to be important for the isolation and structural characterization of epitopes responsible for susceptibility of the gonococcus to killing and thus for the development of a vaccine against invasive gonococcal disease.
Asunto(s)
Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/inmunología , Neisseria gonorrhoeae/clasificación , Pruebas de Aglutinación , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Inmunoensayo , Neisseria gonorrhoeae/inmunología , SerotipificaciónRESUMEN
The antigen-specific basis of human serum immunoglobulin G antibody response to complicated gonococcal infection was studied in 13 patients by using the Western blot technique for transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose paper. Of 13 patients (8 with disseminated gonococcal infection, 4 with pelvic inflammatory disease, 1 with gonococcal epididymitis), 12 reacted with protein I antigens and 9 with lipopolysaccharide (LPS). Sera from eight patients reacted with both protein I and LPS, whereas sera from four reacted only with protein I, and one sera reacted with LPS alone. One serum with antibody to both protein I and LPS by Western blot analysis was tested for bactericidal activity before and after adsorption of antibody to LPS. Removal of antibody to LPS reduced the bactericidal titer of this serum from 1:100 to 1:50, indicating that antibody to both antigens may be bactericidal for Neisseria gonorrhoeae.
Asunto(s)
Formación de Anticuerpos , Epítopos , Gonorrea/inmunología , Inmunoglobulina G/inmunología , Adsorción , Ensayo de Inmunoadsorción Enzimática , Humanos , Lipopolisacáridos/inmunología , MasculinoRESUMEN
antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.
Asunto(s)
Antígenos Bacterianos/análisis , Macrófagos/inmunología , Neisseria gonorrhoeae/inmunología , Proteínas Opsoninas/inmunología , Fagocitosis , Reacciones Antígeno-Anticuerpo , Antígenos de Superficie/análisis , Fimbrias Bacterianas/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Lipopolisacáridos/inmunología , Proteínas de la Membrana/inmunologíaRESUMEN
Net oxygen production during photosynthesis by all plants requires adaptation to intracellular O2 tensions in excess of 0.21 ATA. The symbiotic association of zooxanthellae (algae) in the tissues of many actinozoans and hydrozoans (corals and anemones) suggests such an adaptation in these tissues as well, and raises the question as to degree. Oxygen production by zooxanthellae in a single coral head of Montastrea cavernosum was monitored daily in situ in a closed, recirculating 15-liter system. The net photosynthetic activity repeatedly raised the PO2 to more than 0.5 ATA, indicating that even higher tensions existed in the coral's tissues in order to cause this increase and suggesting that coral tissue may represent another example of an oxygen-adapted tissue.