RESUMEN
Psychological stress is associated with gastrointestinal (GI) distress. This secondary analysis from a randomised, double-blind, placebo-controlled study examined whether three different probiotics could normalise self-reported stress-associated GI discomfort and reduce overall self-reported stress. Undergraduate students (n=581) received Lactobacillus helveticus R0052, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium bifidum R0071, or placebo. Participants self-reported 2 outcomes for a 6-week period, which included final academic exams: daily level of stress (0=no stress to 10=extremely stressed) and weekly three diarrhoea-related symptoms (DS, 1=no discomfort to 7=severe discomfort) using the GI Symptom Rating Scale. Self-reported stress was positively related to DS (P=0.0068). Mean DS scores were lower with B. bifidum versus placebo at week 2 at the average level of stress and the average body mass index (BMI). DS scores were lower with B. bifidum at week 5 versus week 0 and 1 and with B. infantis R0033 at week 6 versus week 0. DS scores were higher when antibiotics were used in the prior week with placebo (P=0.0092). DS were not different with or without antibiotic use with the probiotics. Only B. bifidum had an effect on self-reported stress scores (P=0.0086). The self-reported stress score was also dependent on hours of sleep per day where it decreased by 0.13 for each additional hour of sleep. During a stressful period, B. bifidum R0071 decreases DS and self-reported stress scores. This trial was registered at clinicaltrials.gov as NCT01709825.
Asunto(s)
Bifidobacterium bifidum/inmunología , Diarrea/patología , Diarrea/terapia , Probióticos/administración & dosificación , Estrés Fisiológico , Bifidobacterium longum/inmunología , Método Doble Ciego , Femenino , Humanos , Lactobacillus helveticus/inmunología , Masculino , Placebos/administración & dosificación , Estudiantes , Encuestas y Cuestionarios , Resultado del Tratamiento , Estados UnidosRESUMEN
Our laboratories are developing treadmill-based gait analysis employing sheep to investigate potential efficacy of intra-dural spinal cord stimulation in the treatment of spinal cord injury and neuropathic pain. As part of efforts to establish the performance characteristics of the experimental arrangement, this study measured the treadmill speed via a tachometer, video belt-marker timing and ambulation-rate observations of the sheep. The data reveal a 0.1-0.3% residual drift in the baseline (unloaded) treadmill speed which increases with loading, but all three approaches agree on final speed to within 1.7%, at belt speeds of ≈ 4 km/h. Using the tachometer as the standard, the estimated upper limit on uncertainty in the video belt-marker approach is ± 0.18 km h(-1) and the measured uncertainty is ± 0.15 km h(-1). Employment of the latter method in determining timing differences between contralateral hoof strikes by the sheep suggests its utility in assessing severity of SCI and responses to therapeutic interventions.
Asunto(s)
Prueba de Esfuerzo , Neuralgia/fisiopatología , Traumatismos de la Médula Espinal/fisiopatología , Caminata/clasificación , Caminata/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , OvinosRESUMEN
The aim of the studies was to determine the effects of calcium carbonate and calcium phosphate supplementation on faecal Lactobacillus spp., with and without a probiotic supplement, in healthy adults. Study 1 comprised of a randomised, double-blind, crossover design; participants (n=15) received 2 capsules/d of 250 mg elemental calcium as calcium carbonate (Ca1) and calcium phosphate (Ca2) each for 2-week periods, with 2-week baseline and washout periods. Study 2 was a randomised, double-blind, crossover design; participants (n=17) received 2 capsules/d of Lactobacillus helveticus R0052 and Lactobacillus rhamnosus R0011 (probiotic) alone, the probiotic with 2 capsules/d of Ca1, and probiotic with 2 capsules/d of Ca2 each for 2-week periods with 2-week baseline and washout periods. In both studies, stools were collected during the baseline, intervention and washout periods for Lactobacillus spp. quantification and qPCR analyses. Participants completed daily questionnaires of stool frequency and compliance. In Study 1, neither calcium supplement influenced viable counts of resident Lactobacillus spp., genome equivalents of lactic acid bacteria or stool frequency. In Study 2, faecal Lactobacillus spp. counts were significantly enhanced from baseline when the probiotic was administered with Ca2 (4.83±0.30, 5.79±0.31) (P=0.02), but not with Ca1 (4.98±0.31) or with the probiotic alone (5.36±0.31, 5.55±0.29) (not significant). Detection of L. helveticus R0052 and L. rhamnosus R0011 was significantly increased with all treatments, but did not differ among treatments. There were no changes in weekly stool frequency. Calcium phosphate co-administration may increase gastrointestinal survival of orally-administered Lactobacillus spp.
Asunto(s)
Fosfatos de Calcio/farmacología , Heces/microbiología , Lacticaseibacillus rhamnosus/efectos de los fármacos , Lactobacillus helveticus/efectos de los fármacos , Probióticos/farmacología , Adolescente , Adulto , Estudios Cruzados , Suplementos Dietéticos , Método Doble Ciego , Femenino , Humanos , Lactobacillus helveticus/aislamiento & purificación , Lacticaseibacillus rhamnosus/aislamiento & purificación , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
A probiotic formulation of Enterococcus faecium R0026 and Bacillus subtilis R0179 has been evaluated in previous clinical trials. However, B. subtilis R0179 has not been evaluated as a single probiotic strain or in combination with other strains at doses higher than 0.1×109 cfu. To establish oral dose-response tolerance and gastrointestinal (GI) viability of B. subtilis R0179, a randomised, double-blind, placebo-controlled trial in healthy adults (n=81; 18-50 years old) was conducted. Participants received B. subtilis R0179 at 0.1, 1.0 or 10×109 cfu/capsule/day or placebo for four weeks. General wellness was assessed using a daily questionnaire evaluating GI, cephalic, ear-nose-throat, behavioural, emetic, and epidermal symptoms. GI symptoms were further evaluated using a weekly gastrointestinal symptom rating scale (GSRS). GI transit viability of B. subtilis R0179 was assessed by plating and microbiota analysis by 16S rRNA at baseline, week 4 of the intervention and washout. General wellness and GI function were not affected by oral consumption of B. subtilis R0179 at any dose. Daily questionnaire syndrome scores were not different from baseline and did not exceed a clinically significant score of 1. GSRS syndrome scores were not different from baseline and ranged from 1.1±0.1 to 1.9±0.2. Faecal viable counts of B. subtilis R0179 demonstrated a dose response: the placebo group (1.1±0.1 log10 cfu/g) differed from 0.1×109 (4.6±0.1 log10 cfu/g), 1×109 (5.6±0.1 log10 cfu/g) and 10×109 (6.4±0.1 log10 cfu/g) (P<0.0001). No significant changes in phyla were observed, but sequence reads binned to multiple operational taxonomic units matching closest to Ruminococci increased during probiotic supplementation. B. subtilis R0179 survives passage through the human GI tract and is well tolerated by healthy adults at intakes from 0.1 to 10×109 cfu/day. The trial has been registered at www.clinicaltrials.gov under NCT01802151.
Asunto(s)
Bacillus subtilis/fisiología , Heces/microbiología , Viabilidad Microbiana , Probióticos/administración & dosificación , Probióticos/efectos adversos , Administración Oral , Adulto , Recuento de Colonia Microbiana , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Método Doble Ciego , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Tracto Gastrointestinal/microbiología , Humanos , Placebos/administración & dosificación , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
Evidence-based medicine has emerged as a valuable tool to guide clinical decision-making, by summarizing the best possible evidence for both diagnostic and treatment strategies. Imaging plays a critical role in the evaluation and treatment of patients with acute ischemic stroke, especially those who are being considered for thrombolytic or endovascular therapy. Time from stroke-symptom onset to treatment is a strong predictor of long-term functional outcome after stroke. Therefore, imaging and treatment decisions must occur rapidly in this setting, while minimizing unnecessary delays in treatment. The aim of this review was to summarize the best available evidence for the diagnostic and therapeutic management of patients with acute ischemic stroke.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/terapia , Terapia Trombolítica/estadística & datos numéricos , Tomografía Computarizada por Rayos X/métodos , Ensayos Clínicos como Asunto , Medicina Basada en la Evidencia , Humanos , Prevalencia , Pronóstico , Resultado del TratamientoAsunto(s)
Encefalopatías/genética , Exodesoxirribonucleasas/genética , Mutación/genética , Fosfoproteínas/genética , Enfermedades de la Retina/genética , Vasos Retinianos/patología , Adulto , Encefalopatías/complicaciones , Imagen de Difusión por Resonancia Magnética/métodos , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Enfermedades de la Retina/complicacionesRESUMEN
AIMS: To investigate whether changes in glucose concentrations during an OGTT in obese children reflect the presence of peripheral insulin resistance and/or cardiovascular risk factors more closely than single measurements of fasting plasma glucose (FPG). METHODS: One hundred and twenty-two obese children attending our Paediatric Obesity Service underwent formal OGTTs, following the measurement of blood pressure and fasting levels of insulin, glucose and lipid profiles in the majority. Fasting insulin was used as a surrogate measure of insulin sensitivity. Three different child-specific definitions for metabolic syndrome were used to identify clustering of cardiovascular risk factors in 65 of these children. RESULTS: In the whole group, 10.7% had IGT but changes in glucose during the OGTT were not influenced by age, sex, pubertal status or raw (or age- and sex-adjusted) body mass index (BMI). During the OGTT, FPG, glucose at 60 min and area under the glucose curve correlated highly with fasting insulin. Children with metabolic syndrome (defined using any of three definitions) had comparable FPG levels to those without metabolic syndrome, but they demonstrated significantly elevated glucose levels at 60 min. On sub-group analysis, obese children with normal carbohydrate metabolism were significantly more likely to have a 1 h glucose level > or = 7.8 mmol/l if they had metabolic syndrome (P = 0.026). CONCLUSIONS: These data suggest that an elevated 1 h post-load glucose measurement is seen in obese children who have a coexistent clustering of cardiovascular risk factors.
Asunto(s)
Glucemia/metabolismo , Prueba de Tolerancia a la Glucosa , Síndrome Metabólico/metabolismo , Obesidad/metabolismo , Adolescente , Adulto , Niño , Preescolar , Métodos Epidemiológicos , Femenino , Humanos , Masculino , Síndrome Metabólico/sangreRESUMEN
AIM: To identify clinical features which predict those most at risk of co-morbidities within an obesity clinic. METHODS: Children attending an obesity clinic had fasting glucose, insulin, and lipids measured prior to a standard oral glucose tolerance test (OGTT). History and examination established birth weight, family history of type 2 diabetes/obesity, pubertal status, and presence of acanthosis nigricans. Central and total fat mass was estimated by bio-impedance. RESULTS: Of the 126 children evaluated, 10.3% (n = 13) had impaired glucose tolerance (IGT); the majority (n = 11) of these would not have been identified on fasting glucose alone. Those with IGT were more likely to have a parental history of type 2 diabetes (relative risk 3.5). IGT was not associated with acanthosis nigricans. Twenty five per cent (n = 19) of those evaluated (n = 75) had evidence of the "metabolic syndrome" (MS). HDL cholesterol and triglyceride levels were related to insulin sensitivity (HOMA-R); HDL cholesterol was also related to birth weight SDS. We observed a trend for those with MS to have a lower birth weight SDS. The severity of obesity did not influence the likelihood of IGT or MS. CONCLUSIONS: Significant numbers of obese children have associated co-morbidities. Analysis of fasting blood glucose samples alone is not satisfactory to adequately evaluate glucose homoeostasis. The overall level of obesity does not predict co-morbidities. Special attention should be given to those with parental diabetes and a history of low birth weight who are more likely to have IGT and abnormal lipid profiles respectively.
Asunto(s)
Síndrome Metabólico/etiología , Obesidad/complicaciones , Adolescente , Peso al Nacer , Glucemia/metabolismo , Índice de Masa Corporal , Niño , Preescolar , HDL-Colesterol/sangre , Diabetes Mellitus Tipo 2/genética , Ayuno/sangre , Femenino , Intolerancia a la Glucosa , Prueba de Tolerancia a la Glucosa , Humanos , Lactante , Recién Nacido , Insulina/sangre , Masculino , Síndrome Metabólico/sangre , Obesidad/sangre , Servicio Ambulatorio en Hospital , Triglicéridos/sangreAsunto(s)
Enfermedades de la Coroides/patología , Hematopoyesis Extramedular , Adulto , Coroides/irrigación sanguínea , Coroides/patología , Enfermedades de la Coroides/etiología , Hemorragia del Ojo/etiología , Lesiones Oculares Penetrantes/complicaciones , Humanos , Masculino , Desprendimiento de Retina/etiología , Esclerótica/lesionesRESUMEN
Trypanosoma cruzi (Schyzotrypanum, Chagas, 1909), and Chagas disease are endemic in captive-reared baboons at the Southwest Foundation for Biomedical Research, San Antonio, Texas. We obtained PCR amplification products from DNA extracted from sucking lice collected from the hair and skin of T. cruzi-infected baboons, with specific nested sets of primers for the protozoan kinetoplast DNA, and nuclear DNA. These products were hybridized to their complementary internal sequences. Selected sequences were cloned and sequencing established the presence of T. cruzi nuclear DNA, and minicircle kDNA. Competitive PCR with a kDNA set of primers determined the quantity of approximately 23.9 +/- 18.2 T. cruzi per louse. This finding suggests that the louse may be a vector incidentally contributing to the dissemination of T. cruzi infection in the baboon colony.
Asunto(s)
Vectores de Enfermedades , Infestaciones por Piojos/veterinaria , Papio/parasitología , Phthiraptera/parasitología , Trypanosoma cruzi/aislamiento & purificación , Animales , ADN de Cinetoplasto/análisis , ADN Protozoario/análisis , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa , Trypanosoma cruzi/genéticaRESUMEN
We characterized the development of the anti-centromere antibody in a patient prior to the development of CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) symptoms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting (IgG and IgM) of cellular extracts enriched for centromere antigens and indirect immunofluorescence were used to study the anti-centromere immune response. The sera recognized 3 centromere antigens with molecular masses 18,000 (CENP-A), 50,000 (CENP-D), and 80,000 (CENP-B). For CENP-A, IgM was present before the appearance of the IgG response. Anti-CENP-D revealed an IgM response that decreased over time but no IgG, while CENP-B showed an IgG response that strengthened and then weakened over time. The appearance of an anti-centromere nuclear fluorescence pattern correlated with the appearance of IgG anti-CENP-A. Signs and symptoms typical of CREST began about 4 years after antibodies to centromere antigens were found. The development of the CREST syndrome in our patient was preceded by the appearance of anti-centromere autoantibodies. For at least one of the antigens (CENP-A), there was an immunoglobulin class switch from IgM to IgG.
Asunto(s)
Autoanticuerpos/inmunología , Síndrome CREST/inmunología , Centrómero/inmunología , Lupus Vulgar/inmunología , Síndrome de Sjögren/inmunología , Adulto , Síndrome CREST/complicaciones , Femenino , Humanos , Immunoblotting , Lupus Vulgar/complicaciones , Microscopía Fluorescente , Síndrome de Sjögren/complicacionesRESUMEN
Central nervous system (CNS)-resident macrophages (microglia) normally express negligible or low level MHC class II, but this is up-regulated in graft-vs-host disease (GvHD), in which a sparse CNS T cell infiltrate is observed. Relative to microglia from the normal CNS, those from the GvHD-affected CNS exhibited a 5-fold up-regulation of characteristically low CD45, MHC class II expression was increased 10- to 20-fold, and microglial cell recoveries were enhanced substantially. Immunohistologic analysis revealed CD4+ alphabetaTCR+CD2+ T cells scattered infrequently throughout the CNS parenchyme, 90% of which were blast cells of donor origin. An unusual clustering of activated microglia expressing strongly enhanced levels of CD11b/c and MHC class II was a feature of the GvHD-affected CNS, and despite the paucity of T lymphocytes present, activated microglial cell clusters were invariably intimately associated with these T cells. Moreover, 70% of T cells in the CNS were associated with single or clustered MHC class II+ microglia, and interacting cells were predominantly deep within the tissue parenchyme. Approximately 3.7% of the microglia that were freshly isolated from the GvHD-affected CNS were cycling, and proliferating cell nuclear Ag-positive microglia were detected in situ. Microglia from GvHD-affected animals sorted to purity by flow cytometry and cultured, extended long complex processes, exhibited spineous processes, and were phagocytic and highly motile. These outcomes are consistent with direct tissue macrophage-T cell interactions in situ that lead to activation, proliferation, and expansion of the responding tissue-resident cell.
Asunto(s)
Movimiento Celular/inmunología , Sistema Nervioso Central/inmunología , Activación de Linfocitos , Microglía/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Apoptosis , Adhesión Celular/inmunología , Comunicación Celular/inmunología , División Celular/inmunología , Sistema Nervioso Central/citología , Cruzamientos Genéticos , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Inmunofenotipificación , Activación de Macrófagos , Microglía/citología , Microglía/fisiología , Fagocitos/inmunología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , Bazo/citología , Subgrupos de Linfocitos T/citologíaRESUMEN
Microglia, a type of tissue macrophage, are the only cells in the central nervous system (CNS) parenchyma to express some major histocompatibility complex (MHC) class II constitutively or to upregulate expression readily. They are thought to play a role in CD4 T cell activation in autoimmune diseases such as multiple sclerosis, as well as in neurodegenerative conditions, Alzheimer's disease in particular. We show here that highly purified MHC class II+ microglia when tested directly ex vivo do indeed support an effector response by an encephalitogenic myelin basic protein-reactive CD4 T cell line from which production of the proinflammatory cytokines, interferon gamma and tumor necrosis factor, is elicited, but not interleukin (IL)-2 secretion or proliferation. After this interaction, the T cells die by apoptosis. Other nonmicroglial but CNS-associated macrophages isolated in parallel stimulate full T cell activation, including IL-2 production, proliferation, and support T cell survival. Neither CNS-derived population expresses B7.1/B7.2. Resident macrophages that terminate effector T cells in tissues constitute a novel and broadly applicable regulatory measure of particular relevance to processes of self-tolerance against sequestered antigens.
Asunto(s)
Apoptosis , Linfocitos T CD4-Positivos/inmunología , Activación de Linfocitos , Macrófagos/inmunología , Microglía/inmunología , Animales , Células Presentadoras de Antígenos , Antígenos CD/análisis , Enfermedades Autoinmunes/etiología , Antígeno B7-1/análisis , Antígeno B7-2 , Linfocitos T CD4-Positivos/efectos de los fármacos , Sistema Nervioso Central/citología , Anergia Clonal , Enfermedad Injerto contra Huésped , Antígenos de Histocompatibilidad Clase II , Interferón gamma/biosíntesis , Interleucina-2/farmacología , Antígenos Comunes de Leucocito , Glicoproteínas de Membrana/análisis , Proteína Básica de Mielina/inmunología , Ratas , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Dispase was used to digest central nervous system (CNS) tissue for isolation of the fixed tissue macrophage population (CNS microglia) and other leucocytes present. An apparent reduction in expression of some leucocyte cell surface markers was investigated further by addition of CD45b allotype-marked post-activated CD4+ T cells of known phenotype to CNS tissue preparations derived from a CD45a-expressing rat strain, before enzymatic treatment. Assessment of these T cells for expression of a range of surface molecules after completion of the isolation procedure revealed almost complete loss of expression of CD4 and CD25 and reduced expression of a number of other surface antigens.
Asunto(s)
Antígenos CD/análisis , Endopeptidasas/farmacología , Macrófagos/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD4/análisis , Femenino , Inmunofenotipificación , Antígenos Comunes de Leucocito/análisis , Masculino , Ratas , Ratas Endogámicas Lew , Receptores de Interleucina-2/análisisRESUMEN
The critical role of tumor necrosis factor (TNF) as a mediator in autoimmune inflammatory processes is evident from in vivo studies with TNF-blocking agents. However, the mechanisms by which TNF, and possibly also its homologue lymphotoxin alpha, contributes to development of pathology in rheumatoid arthritis and Crohn disease and in animal models like experimental autoimmune encephalomyelitis is unclear. Possibilities include regulation of vascular adhesion molecules enabling leukocyte movement into tissues or direct cytokine-mediated effector functions such as mediation of tissue damage. Here we show that administration of a TNF receptor (55 kDa)-IgG fusion protein prevented clinical signs of actively induced experimental autoimmune encephalomyelitis. Significantly, the total number of CD4+ T lymphocytes isolated from the central nervous system of clinically healthy treated versus diseased control animals was comparable. By using a CD45 congenic model of passively transferred experimental autoimmune encephalomyelitis to enable tracking of myelin basic protein-specific effector T lymphocytes, prevention of clinical signs of disease was again demonstrated in treated animals but without quantitative or qualitative impediment to the movement of autoreactive T lymphocytes to and within the central nervous system. Thus, despite the uninterrupted movement of specific T lymphocytes into the target tissue, subsequent disease development was blocked. This provides compelling evidence for a direct effector role of TNF/lymphotoxin alpha in autoimmune tissue damage.
Asunto(s)
Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Receptores del Factor de Necrosis Tumoral/química , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Movimiento Celular , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Inmunidad Celular , Antígenos Comunes de Leucocito/análisis , Masculino , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes de FusiónRESUMEN
AIMS: This study aimed to isolate and classify by flow cytometry, the cell surface phenotype of microglia in the normal rat retina with a view to identifying putative antigen presenting cells (APC) within the retina, which has to date not been possible by immunohistochemistry. METHODS: Normal rat retinal microglia were isolated and classified using a modification of an isolation technique employing graduated Percoll density gradient cell separation and flow cytometric phenotypic criteria used for CNS microglia. RESULTS: Retinal microglia can be defined by flow cytometry on the basis of their CD45lowCD11b/c+CD4low cell surface expression. Constitutive MHC class II expression in the normal rat retina was confined almost exclusively to a very minor population of cells expressing neither low (microglia) nor high levels of CD45. Three colour flow cytometric analysis confirmed that these MHC class II positive cells were ED2+. CONCLUSIONS: Using this sensitive isolation technique we have identified the cell surface characteristics of ramified, resident microglia, and found that they do not constitutively express MHC class II. There is, however, constitutive MHC class II expression on a phenotypically distinct population of cells (CD45low/highED2+). We propose these cells are the counterpart of the perivascular macrophages found in the CNS which present antigen to extravasating T cells, although their exact retinal location can only be confirmed by immunohistochemical analysis. The role of parenchymal microglia as APC remains undefined. Future isolation of microglia and putative perivascular cells using this technique will help identify the role these cells play in the initiation and perpetuation of immune responses within the retina.
Asunto(s)
Antígenos Comunes de Leucocito/análisis , Retina/inmunología , Animales , Antígenos CD4/análisis , Sistema Nervioso Central/inmunología , Citometría de Flujo , Integrina alfaXbeta2/análisis , Antígeno de Macrófago-1/análisis , Complejo Mayor de Histocompatibilidad/inmunología , Microglía/inmunología , Fenotipo , Ratas , Ratas Endogámicas LewRESUMEN
Ramified microglia in the adult central nervous system (CNS) are the principal glial element up-regulating MHC class I and II expression in response to inflammatory events or neuronal damage. A proportion of these cells also express MHC class II constitutively in the normal CNS. The role of microglia as APCs for CD4+ T cells extravasating into the CNS remains undefined. In this study, using irradiation bone marrow chimeras in CD45-congenic rats, the phenotype CD45lowCD11b/c+ is shown to identify microglial cells specifically within the CNS. Highly purified populations of microglia and nonmicroglial but CNS-associated macrophages (CD45highCD11b/c+) have been obtained directly from the adult CNS, by using flow cytometric sorting. Morphologically, freshly isolated microglia vs other CNS macrophages are quite distinct. Of the two populations recovered from the normal CNS, it is the minority CD45highCD11b/c+ transitional macrophage population, and not microglia, that is the effective APC for experimental autoimmune encephalomyelitis-inducing CD4+ myelin basic protein (MBP)-reactive T cells. CD45highCD11b/c+ CNS macrophages also stimulate MBP-reactive T cells without addition of MBP to culture, suggesting presentation of endogenous Ag. This is the first study in which microglia vs other CNS macrophages have been analyzed for APC ability directly from the CNS, with substantial cross-contamination between the two populations eliminated. The heterogeneity of these populations in terms of APC function is clearly demonstrated. Evidence is still lacking that adult CNS microglia have the capacity to interact with and stimulate CD4+ T cells to proliferate or secrete IL-2.
Asunto(s)
Separación Celular/métodos , Sistema Nervioso Central/inmunología , Citometría de Flujo/métodos , Macrófagos/inmunología , Microglía/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Células Presentadoras de Antígenos/inmunología , Antígenos CD11/inmunología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Sistema Nervioso Central/citología , Femenino , Inmunofenotipificación/métodos , Interleucina-2/biosíntesis , Antígenos Comunes de Leucocito/biosíntesis , Activación de Linfocitos/inmunología , Macrófagos/citología , Masculino , Microglía/citología , Quimera por Radiación/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
Metatherian (marsupial) mammals possess a non-random form of X-chromosome inactivation in which the paternally-derived X is always the one inactivated. To examine the progression of X-linked gene expression during metatherian development, we compared relative levels of the maternally and paternally encoded Gpd gene products in heterozygous female Virginia opossums (Didelphis virginiana) across a major portion of the developmental period. Panels of tissues obtained from fetuses, newborns, and pouch young were examined via polyacrylamide gel electrophoresis of the G6PD protein. As in adults, G6PD phenotypes in these developmental stages were highly skewed in favor of the maternal allele product, but in some tissues there was a marked increase in paternal allele expression with advancing developmental age. However, even by 42 days of post-partum development, expression of the paternal Gpd allele had not attained the adult, tissue-specific activity pattern. Our findings indicate remarkable developmental changes in the activity of the paternal allele in several tissues/organs continuing well into mid pouch-life stages and beyond. Specifically we found that 1) a substantially repressed paternal Gpd gene is present in the cells of female stage 29 fetuses and later developmental stages, 2) the activity state of the paternal Gpd gene is not fixed during early embryonic development in this species, 3) major changes in paternal Gpd expression occur in advanced developmental stages and comprise a maturation of the gene expression pattern during ontogeny, and 4) alterations of paternal Gpd allele activity during development occur in a tissue-specific manner.
Asunto(s)
Compensación de Dosificación (Genética) , Glucosafosfato Deshidrogenasa/genética , Zarigüeyas/genética , Alelos , Animales , Animales Recién Nacidos , Diploidia , Femenino , Feto , Expresión Génica , Masculino , Zarigüeyas/embriología , FenotipoRESUMEN
Peripheral nerves are frequently damaged during infection with Mycobacterium leprae. Although Schwann cells are host for this obligate intracellular parasite, the mechanisms of immunopathology are unresolved. This study examines the ability of Lewis rat Schwann cells to present an exogenous Mycobacterium leprae protein, the heat shock protein 70 (hsp70), to antigen-specific T lymphocytes isolated from the lymph nodes of immunised rats. Secondary reactivation of hsp70-specific T lymphocytes occurred producing an antigen-specific lymphoproliferative response. This was inhibited by monoclonal antibodies against rat major histocompatibility complex (MHC) class II molecules, but not antibodies against MHC class I molecules. Coculture of Schwann cells with the M.leprae hsp70-specific T lymphocytes and antigen (MLrp70) induced the expression of MHC class II molecules on the Schwann cell's surface. Although M.leprae hsp70 is immunodominant in the host response to the bacillus, there is a high degree of homology between human and M.leprae hsp70. The M.leprae hsp70-specific T lymphocytes also recognised human hsp70 presented by Schwann cells confirming that antigenic determinants are conserved between the proteins. The ability of Schwann cells to present protein antigens in an MHC class II-restricted manner, to antigen-specific T lymphocytes involved in surveillance of the peripheral nervous system, may play an important role in the activation of an immunological reaction associated with nerve damage seen in tuberculoid leprosy.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Proteínas de Choque Térmico/inmunología , Mycobacterium leprae/inmunología , Células de Schwann/inmunología , Linfocitos T/inmunología , Animales , Células Cultivadas , Epítopos , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Cinética , Activación de Linfocitos , Ratas , Ratas Endogámicas LewRESUMEN
We describe a protocol to cast nondenaturing polyacrylamide gradient gels (SFBR3/31) for the size resolution of lipoproteins. The protocol yields gels with minimal lot-to-lot variation in length and electrophoretic properties. Absorbance profiles of cholesterol-stained lipoproteins in baboon sera were used to estimate the relative amounts of stain in four lipoprotein size classes (VLDL+LDL, HDL1, HDL2, and HDL3). When compared with gels from a commercial source, the SFBR3/31 gels gave very similar results in terms of precision (coefficients of variation) and of estimated amounts of lipoproteins in the four size classes. In other studies, we estimated peak diameters of protein-stained human lipoproteins after calibrating the gels with size standards. Peak diameters estimated using SFBR3/31 gels were highly correlated (r2 = 0.99, n = 33) with those estimated using gels from a commercial source. We conclude that the protocol reliably produces gradient gels that are suitable for the analysis of lipoprotein phenotypes.