RESUMO
Patients with primary Sjögren's syndrome (pSS) have an increased risk of developing lymphomas, particularly the subtype mucosa-associated lymphoid tissue (MALT) lymphoma. Chronic antigen stimulation and increased activation of nuclear factor-κB (NF-κB) are important factors for the pathogenesis of MALT lymphomas. Protein A20 is an inhibitor of NF-κB. A recent study of pSS-associated MALT lymphomas identified potential functional abnormalities in the TNFAIP3 gene, which encodes protein A20. The present study aimed to assess protein A20 by immunohistochemistry (IHC) in minor salivary glands (MSGs) and lymphoma tissue sections of patients with pSS and investigate a potential association with lymphoma development. Protein A20 staining in lymphocytes was scored in four categories (0 = negative, 1 = weak, 2 = moderate and 3 = strong). For statistical purposes, these scores were simplified into negative (scores 0-1) and positive (scores 2-3). We investigated associations between protein A20-staining, focus scores, germinal centre (GC)-like structures and monoclonal B-cell infiltration in MSGs. MSG protein A20 staining was weaker in pSS patients with lymphomas than in those without lymphomas (P = 0.01). Weak protein A20 staining was also highly associated with a lack of GC formation (P < 0.01). Finally, weaker A20 staining was observed in the majority of pSS-associated MALT lymphoma tissues. In conclusion, we found absent or weak protein A20 immunoreactivity in MSGs of patients with pSS with lymphomas. This finding indicates that protein A20 downregulation in lymphocytes might be a mechanism underlying lymphoma genesis in patients with pSS.
Assuntos
Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfoma de Zona Marginal Tipo Células B/epidemiologia , Proteínas Nucleares/metabolismo , Glândulas Salivares/patologia , Síndrome de Sjogren/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Feminino , Centro Germinativo/patologia , Humanos , Imuno-Histoquímica , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Síndrome de Sjogren/diagnóstico , Proteína 3 Induzida por Fator de Necrose Tumoral alfaRESUMO
Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/patologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/mortalidade , Linfócitos do Interstício Tumoral/imunologia , Metástase Neoplásica/diagnóstico , Metástase Neoplásica/patologia , Estadiamento de Neoplasias , Prognóstico , Índice de Gravidade de Doença , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: An association between atopic disease and gastrointestinal complaints has been suggested. AIM: To explore the association between atopic disease, gastrointestinal symptoms, and possible gastrointestinal manifestations of atopic disease in patients with self-reported food hypersensitivity. METHODS: Symptoms, skin prick tests, serum markers of allergy and intestinal permeability were recorded in 71 adult patients. Eosinophils, tryptase- and IgE-positive cells were counted in duodenal biopsies. RESULTS: Sixty-six (93%) patients had irritable bowel syndrome (IBS) and 43 (61%) had atopic disease, predominantly rhinoconjunctivitis. All 43 were sensitized to inhalant allergens, 29 (41%) to food allergens, but food challenges were negative. Serum total IgE and duodenal IgE-positive cell counts were significantly correlated (P < 0.0001) and both were significantly higher in atopic than in non-atopic patients (P < 0.0001 and P = 0.003 respectively). IgE-positive cells appeared to be 'armed' mast cells. Intestinal permeability was significantly elevated in atopic compared with non-atopic patients (P = 0.02). Gastrointestinal symptoms and numbers of tryptase-positive mast cells and eosinophils did not differ between groups. CONCLUSIONS: Patients with self-reported food hypersensitivity had a high prevalence of IBS and atopic disease. Atopic patients had increased intestinal permeability and density of IgE-bearing cells compared with non-atopic patients, but gastrointestinal symptoms did not differ between groups.
Assuntos
Gastroenteropatias/complicações , Imunoglobulina E/imunologia , Doenças Inflamatórias Intestinais/complicações , Adolescente , Adulto , Idoso , Alérgenos/imunologia , Biomarcadores/sangue , Feminino , Hipersensibilidade Alimentar/complicações , Hipersensibilidade Alimentar/epidemiologia , Hipersensibilidade Alimentar/patologia , Gastroenteropatias/epidemiologia , Gastroenteropatias/patologia , Humanos , Imunoglobulina E/metabolismo , Doenças Inflamatórias Intestinais/epidemiologia , Doenças Inflamatórias Intestinais/patologia , Masculino , Pessoa de Meia-Idade , Prevalência , Testes Cutâneos/métodos , Adulto JovemRESUMO
Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following (14)C-gamma-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.
Assuntos
Microssomos Hepáticos/efeitos dos fármacos , Saponinas/farmacologia , Animais , Anticoagulantes , Soluções Tampão , Dióxido de Carbono/química , Radioisótopos de Carbono , Fracionamento Químico , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Octoxinol , Permeabilidade , Polietilenoglicóis , Precursores de Proteínas/metabolismo , Protrombina/química , Protrombina/metabolismo , Ratos , Ratos Wistar , Vitamina K/metabolismo , VarfarinaRESUMO
Previous studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR beta-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR beta-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8alphaalpha and CD8alphabeta single-positive (SP) IELs used most Vbeta genes, but relative Vbeta usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vbeta17 and Vbeta19; these two genes accounted collectively for more than half the Vbeta repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping beta-chain repertoires. In conclusion, our results show that rat TCR-alphabeta+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vbeta usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.
Assuntos
Antígenos CD4/análise , Antígenos CD8/análise , Mucosa Intestinal/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/imunologia , Animais , Feminino , RatosAssuntos
Sistema Digestório/citologia , Mucosa Intestinal/citologia , Linfócitos T/citologia , Animais , Doença Celíaca/patologia , Sistema Digestório/imunologia , Humanos , Síndromes de Imunodeficiência/patologia , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/imunologia , Fenótipo , Linfócitos T/imunologiaRESUMO
Recent studies in mice and humans have provided evidence for regional specialization of gut intraepithelial lymphocytes (IEL). Here the authors report striking regional variability in the composition of IEL in rat small and large intestine. Two-colour immunofluorescence in situ analysis showed that the distribution of the CD3+ and CD3- IEL subpopulations varied, the proportion of T cells (CD3+) being higher in the ileum than in the jejunum and smallest in the colon. These differences were explained by variable numbers of the T-cell receptor (TCR)alpha/beta + (both CD8+ and CD4+) but not the TCR gamma/delta + subset. Moreover, the various IEL subpopulations showed distinct intraepithelial distribution patterns with CD4+ and CD8 alpha beta + T cells situated near the lamina propria, while CD3- IEL were located preferentially towards the adluminal part of the epithelium. Regional phenotypic variation did not depend on intestinal colonization because analogous results were obtained in germ-free rats. Conventionalization nevertheless caused a marked relative increase of small intestinal TCR alpha/beta + but not TCR gamma/delta + IEL. This increase was more sustained in the jejunum than ileum and eventually reduced the phenotypic IEL differences between the two sites. By contrast, microbial colonization of the colon induced only a transient increase of intraepithelial TCR alpha/beta + cells with no permanent phenotypic alterations. Both CD3+ and CD3- IEL contained subpopulations that expressed NKR-P1 independent of intestinal colonization. These results demonstrate phenotypic specialization of IEL at different levels of the gut and suggest that the indigenous flora is not essential to this end.
Assuntos
Células Epiteliais/imunologia , Intestino Delgado/imunologia , Intestino Delgado/microbiologia , Lectinas Tipo C , Subpopulações de Linfócitos/classificação , Animais , Antígenos de Superfície/biossíntese , Complexo CD3/metabolismo , Células Epiteliais/classificação , Epitélio/imunologia , Epitélio/microbiologia , Vida Livre de Germes , Íleo/citologia , Íleo/imunologia , Íleo/microbiologia , Imunofenotipagem , Intestino Delgado/citologia , Jejuno/citologia , Jejuno/imunologia , Jejuno/microbiologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/metabolismo , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Ratos , Ratos Endogâmicos Lew , Receptores Imunológicos/biossínteseRESUMO
A rapid and highly sensitive chromogenic microplate assay for quantification of rat and human prothrombin in subcellular fractions and large series of plasma samples has been developed. The assay is based on the conversion of prothrombin to thrombin, using Echis carinatus venom as an activator, and the subsequent cleavage of a chromogenic thrombin specific substrate, D-cyclohexylglycyl-L-alanyl-L-arginine-p-nitroanilide dihydroacetate. para-Nitroaniline being released by the cleavage is then measured at 410 nm with a microplate reader. The method is suitable for analyses of a large number of samples in a short time, measuring prothrombin in the nanogram range (0.3-2.4 ng/40 microliters of sample).
Assuntos
Compostos Cromogênicos , Protrombina/análise , Animais , Humanos , Masculino , Protrombina/efeitos dos fármacos , Protrombina/metabolismo , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Especificidade por Substrato , Trombina/efeitos dos fármacos , Trombina/metabolismo , Venenos de Víboras/metabolismo , Venenos de Víboras/farmacologia , Varfarina/farmacologiaRESUMO
The epithelial glycoprotein called secretory component (SC) is quantitatively the most important receptor of the immune system because it is responsible for external transport of locally produced polymeric IgA (pIgA) to generate remarkably large amounts of secretory IgA. Antibodies of this type constitute the major mediators of specific humoral immunity. Transmembrane SC belongs to the Ig supergene family and functions as a common pIg receptor, also translocating pentameric IgM externally to form secretory IgM. The B cells responsible for mucosal pIg production are initially stimulated in organized mucosa-associated lymphoepithelial structures, particularly the Peyer's patches in the distal small intestine; from these inductive site they migrate as memory cells to exocrine tissues all over the body. Mucous membranes are thus furnished with secretory antibodies in an integrated way, ensuring a variety of specificities at every secretory effector site. There is currently great interest in exploiting this integrated or "common" mucosal immune system for oral vaccination against pathogenic infectious agents and also to induce tolerance in T cell-mediated autoimmune diseases. However, much remains to be learned about mechanisms for antigen uptake and processing necessary to elicit stimulatory or suppressive mucosal immune responses. Moreover, evidence is emerging for the existence of considerable regionalization with regard to functional links between inductive sites and effecter sites of mucosal immunity.
Assuntos
Imunidade nas Mucosas , Componente Secretório/fisiologia , Animais , Formação de Anticorpos , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Genes de Imunoglobulinas , Humanos , Imunoglobulina M/imunologia , Mucosa Intestinal/imunologia , Modelos Imunológicos , Família Multigênica , Nódulos Linfáticos Agregados/imunologia , Componente Secretório/genéticaRESUMO
Previous studies in congenitally athymic nude rats have suggested that the thymus is important for the development of intestinal T cells. Here we have examined the effect of the nude mutation on intraepithelial lymphocyte (IEL) development from the perinatal period. By immunohistochemistry it was shown that CD3(-)CD8 alpha alpha + putative IEL precursors colonized the epithelium of both normal and athymic neonatal rats. Mature T cells, however, did not develop in athymic neonates. In normal rats, gamma delta T cells were present at birth and alpha beta T cells appeared within 8 days of postnatal life. At this age, the composition and relative number of intraepithelial T cells were similar to that in normal adult rats, with the exception that most neonatal T-cell receptor-gamma delta + and -alpha beta + IEL expressed CD8 beta. By contrast, extrathymic T-cell maturation in the gut of congenitally athymic rats occurred slowly, as CD3+ IEL did not appear until 4-6 months of age. These intraepithelial T cells displayed variable phenotypes and appeared to be induced by environmental antigens as they were not found in isolator-kept old nudes. In conclusion, the present results indicate that the major colonization of the gut epithelium with gamma delta and alpha beta T cells expressing CD8 alpha beta takes place perinatally and requires the presence of the thymus. The developmental relationship between these neonatal T cells and more immature CD3- CD8 alpha alpha +/- IEL remains elusive.
Assuntos
Mucosa Intestinal/imunologia , Linfonodos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Antígenos de Linfócitos T gama-delta/análise , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Envelhecimento/imunologia , Animais , Animais Recém-Nascidos , Linfócitos T CD8-Positivos/imunologia , Epitélio/imunologia , Técnicas Imunoenzimáticas , Jejuno/imunologia , Ratos , Ratos NusRESUMO
Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ-free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T-cell receptor (TCR) alpha/beta + IEL in that the proportions of CD4+ and CD8 alpha beta + TCR alpha/beta + cells were increased, while the double negative (CD4- CD8-) fraction was reduced. microbial colonization also influenced the TCR V beta repertoire of CD8+ IEL in that the proportions of V beta 8.2+ and V beta 10+ cells were increased, whereas V beta 8.5+ and V beta 16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same V beta subsets. Three-colour flow-cytometric analysis demonstrated that skewing of the V beta repertoire was most pronounced in the CD8 alpha alpha + subset, although the numerical increase of IEL mainly included the CD8 alpha beta + subset. In contrast to IEL, the TCR V beta repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCR alpha/beta + IEL subpopulations respond dynamically to the microbial gut flora and suggest that their V beta repertoire can be shaped by luminal microbial antigens.
Assuntos
Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citometria de Fluxo , Vida Livre de Germes , Imunidade nas Mucosas , Ativação Linfocitária , Masculino , Mesentério/imunologia , Ratos , Ratos EndogâmicosRESUMO
A simple and highly sensitive chromogenic microplate assay for quantification of rat and human plasminogen in plasma samples and subcellular fractions has been developed. The assay is based on a conversion of plasminogen to plasmin, using urokinase as an activator, and a subsequent cleavage of a chromogenic plasmin substrate D-alanyl-L-cyclohexylalanyl-L-lysine-p-nitroanilide-dihydroacet ate. p-Nitroaniline being released by the cleavage is then measured at 410 nm with a microplate reader. The assay includes an acidification step to make plasminogen more readily activated to plasmin. The method is suitable for analyses of a large number of samples, measuring plasminogen in the nanogram range (0.5-50 ng/50 microliters of sample).
Assuntos
Plasminogênio/análise , Animais , Compostos Cromogênicos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Microquímica/métodos , Concentração Osmolar , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
To study the effects of changed synthesis on intracellular transport of secretory proteins, an acute phase response was induced in rats. The synthesis and secretion of haptoglobin, complement C3, transferrin and albumin were then investigated by pulse labeling with [3H]leucine. Maximal increase in the syntheses of the positive acute phase proteins was observed after 24 h, amounting to an increase of nine, three and twofold for haptoglobin, C3 and transferrin, respectively. The synthesis of albumin decreased to a minimum after 48 h, reaching approximately one fourth of normal synthesis. The time courses for transit through rough endoplasmic reticulum and for secretion were determined after 36 h, and were found to be roughly unchanged for all four proteins despite the different changes in synthesis. The fraction of haptoglobin associated with the microsomal membrane was reduced during the acute phase response, but there was no significant change in membrane association as a function of time after labeling with [3H]leucine. It is concluded that the altered protein synthesis during an acute phase response in vivo has little effect on the time course of secretion of the proteins studied. Furthermore, the basal mechanisms for intracellular transport appear relatively unchanged during this condition.
Assuntos
Reação de Fase Aguda/fisiopatologia , Complemento C3c/metabolismo , Haptoglobinas/metabolismo , Fígado/metabolismo , Albumina Sérica/metabolismo , Transferrina/metabolismo , Animais , Transporte Biológico , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Serum samples from 26 monozygotic twin pairs concordant or discordant with regard to inflammatory bowel disease, and rectal biopsies from 42 twins of the same subject group, were examined for IgG subclasses. They were all compared with normal controls. Almost all affected twins were in clinical remission. Paired immunofluorescence staining of the rectal mucosa showed that those with ulcerative colitis had a significantly higher (p < 0.01) proportion of IgG1 producing mucosal immunocytes than normal controls (78.1% v 55.9%). Conversely, the IgG2 cell fraction was significantly reduced (15.9% v 34.6%). Healthy twins from ulcerative colitis pairs tended to show a raised proportion of IgG1 cells and the IgG2 cell fraction was significantly reduced (p < 0.05). In discordant ulcerative colitis twin pairs, no difference appeared in the cellular IgG subclass pattern between healthy and affected twins. Furthermore, the proportion of IgG1 in these healthy and diseased twins showed good correlation (T = 0.867). The results in rectal mucosa of twins with Crohn's disease were widely scattered and affected twins did not differ significantly from normal controls. Healthy twins, however, showed a marginally raised IgG1 cell proportion, but no correlation was seen between the IgG subclass fractions in discordant Crohn's disease twin pairs. The serum concentrations of IgG1 and IgG2 did not differ from normal controls in twins of either category. These results suggested that in ulcerative colitis, the aberrant mucosal production of IgG1 and IgG2 does not depend on active disease, but is apparently at least partially explained by a genetic impact. Conversely, the mucosal IgG subclass pattern in Crohn's disease appears to be determined mainly by exogenous variables.
Assuntos
Doenças em Gêmeos , Imunoglobulina G/análise , Doenças Inflamatórias Intestinais/imunologia , Mucosa Intestinal/imunologia , Gêmeos Monozigóticos , Adulto , Idoso , Colite Ulcerativa/imunologia , Colo/imunologia , Doença de Crohn/imunologia , Feminino , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Reto/imunologiaRESUMO
The rates of secretion of complement C3, haptoglobin and plasminogen have been determined after pulse labelling with [3H]leucine, and compared to the secretion of prothrombin, albumin and transferrin investigated previously (Kvalvaag, A.H., Tollersrud, O.K. and Helgeland, L. (1988) Biochim. Biophys. Acta 937, 319-327). To study membrane association, rough microsomes were treated with increasing concentrations of saponin, sodium deoxycholate or Triton X-100. All six proteins were quantitated in the soluble and membrane fraction by enzyme immunoassays. At concentrations of saponin from 0.08% to 0.32%, each secretory protein showed a characteristic distribution, almost identical to that obtained with 0.05% sodium deoxycholate or 0.08% Triton X-100. Albumin and transferrin with half-times for secretion (t1/2) 30 and 75 min, respectively, are both almost exclusively found in the luminal fraction (greater than 95%). Prothrombin and plasminogen, which both show an intermediate t1/2 (approx. 55 min), are partially associated with the membranes, as only about 60% was released. Haptoglobin and complement C3 also show some association with the membranes (80-85% released). C3 is secreted at the same rate as prothrombin and plasminogen (t1/2 = 55 min), whereas haptoglobin is secreted more rapidly (t1/2 = 40 min). Accordingly, no correlation between kinetics of secretion and membrane association was demonstrated.
Assuntos
Proteínas Sanguíneas/biossíntese , Membranas Intracelulares/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Complemento C3/biossíntese , Complemento C3/metabolismo , Detergentes , Ensaio de Imunoadsorção Enzimática , Haptoglobinas/biossíntese , Haptoglobinas/metabolismo , Masculino , Plasminogênio/biossíntese , Plasminogênio/metabolismo , Testes de Precipitina , Protrombina/biossíntese , Protrombina/metabolismo , Ratos , Ratos Endogâmicos , Albumina Sérica/biossíntese , Albumina Sérica/metabolismo , Transferrina/biossíntese , Transferrina/metabolismoRESUMO
Plasma from rats with acute inflammatory response was fractionated on Blue Sepharose CL 6B, to separate haptoglobin from albumin and lipoproteins. Affinity chromatography on Blue Sepharose proved to be a convenient method for crude fractionation of plasma. Pure haptoglobin was obtained by the subsequent affinity chromatography on a rabbit-haemoglobin Sepharose column. Minor amounts of rabbit haemoglobin co-eluted from the haemoglobin Sepharose column but did not influence the monospecificity of the antiserum raised in rabbits. By use of the antiserum in single radial immunodiffusion, the concentration of haptoglobin in plasma from normal rats was measured to be 0.5 g/l.
Assuntos
Haptoglobinas/isolamento & purificação , Soros Imunes , Reação de Fase Aguda , Animais , Cromatografia de Afinidade/métodos , Eletroforese em Gel de Poliacrilamida , Haptoglobinas/análise , Haptoglobinas/imunologia , Soros Imunes/imunologia , Imunoeletroforese Bidimensional , Masculino , Ratos , Ratos EndogâmicosRESUMO
An enzyme linked immunoassay (ELISA) for quantitation of rat prothrombin, based on a biotin-streptavidin alkaline phosphatase system is described. The assay utilizes rabbit antiserum raised against purified rat prothrombin. The assay was twenty fold more sensitive than a rat prothrombin assay based on amidolytic activity following activation by Echis carinatus venom. Results obtained with the two assays show good correlation. The ELISA is a valuable tool for quantitation of minute amounts of prothrombin in subcellular fractions and large series of plasma samples.
Assuntos
Ensaio de Imunoadsorção Enzimática , Microssomos Hepáticos/química , Protrombina/análise , Ratos/sangue , Fosfatase Alcalina , Animais , Proteínas de Bactérias , Biotina , Endopeptidases , Imunoglobulina G/imunologia , Coelhos , Ratos Endogâmicos/sangue , Sensibilidade e Especificidade , EstreptavidinaRESUMO
The subclass distribution of IgG-producing immunocytes was examined by immunohistochemistry in nasal and rectal mucosa of infection-prone patients with untreated IgG subclass deficiencies. Biopsy specimens from the two sites were obtained in 18 clinically and serologically well-characterized adult subjects; only a nasal or rectal sample was available from nine similar patients. Chronic lung disease was common in the patient groups with selective serum IgG1 deficiency and combined IgG1 and IgG3 deficiency, whereas the other categories of patients had mainly upper airway and other mild infections. Serum IgG2 or IgG3 deficiency was usually expressed also at the cellular level in rectal mucosa, and the proportion of rectal IgG1 cells was significantly correlated with the IgG1 level (r = 0.90, P less than 0.001). Likewise, there tended to be a decreased expression of the actual subclass at the cellular level in nasal mucosa of patients with serum IgG1 or IgG2 deficiency. Conversely, the median nasal proportion of IgG3 cells was remarkably unaffected by a deficiency of this subclass in serum and rectal mucosa. Interestingly, these patients rather tended to have raised IgG3 and reduced IgG2 cell proportions in their nasal mucosa, although this apparent local IgG3 compensation was nevertheless strongly correlated with the serum IgG3 level (r = 0.87, P less than 0.002). These disparities may reflect different antigenic and mitogenic exposure of the two tissue sites; for example, a persistent protein bombardment of the nasal mucosa that could conceivably override locally a B cell maturation defect. The possible clinical consequences of such variable mucosal expression of IgG subclass deficiencies remain to be studied.
Assuntos
Células Produtoras de Anticorpos/metabolismo , Disgamaglobulinemia/imunologia , Deficiência de IgG , Isotipos de Imunoglobulinas/metabolismo , Mucosa Intestinal/imunologia , Mucosa Nasal/imunologia , Adulto , Idoso , Disgamaglobulinemia/complicações , Feminino , Imunofluorescência , Humanos , Imunidade , Imuno-Histoquímica , Mucosa Intestinal/citologia , Pneumopatias/complicações , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/citologia , Reto/citologia , Reto/imunologiaRESUMO
The steady-state concentration of abnormal plasma prothrombin in warfarin-treated rats (10 mg/kg) was found to be approx. 6% of the plasma prothrombin level in normal rats. The clearance of abnormal plasma prothrombin in warfarin-treated rats was studied using either cycloheximide, to inhibit the synthesis, or vitamin K, to block the appearance of abnormal prothrombin in plasma. The clearance of abnormal plasma prothrombin corresponded to a half-life of approx. 6 h, which is similar to the half-life of normal plasma prothrombin. The de novo synthesis of prothrombin in warfarin-treated and normal rats was compared by measuring the incorporation of [3H]leucine into plasma prothrombin 90 min after an intravenous injection of the isotope. In warfarin-treated rats, accumulated prothrombin precursor was carboxylated and transported into circulation by injecting vitamin K 30 min after isotope administration. On comparing the incorporation of [3H]leucine into plasma prothrombin in warfarin-treated and normal rats, no significant difference in the de novo synthesis was detected. Our results suggest that the secretion of prothrombin in warfarin-treated rats is decreased to 6% of the normal rate. As the de novo synthesis is not affected by warfarin treatment, more than 90% of the newly synthesized prothrombin appears to be degraded intracellularly.
Assuntos
Protrombina/biossíntese , Varfarina/farmacologia , Animais , Cicloeximida/farmacologia , Meia-Vida , Leucina/farmacocinética , Masculino , Matemática , Protrombina/farmacocinética , Ratos , Ratos EndogâmicosRESUMO
The intracellular transport of prothrombin in rat has been studied and compared with the transport of albumin and transferrin. The proteins were immunoisolated from plasma samples after pulse labelling with [3H]leucine and the secretion kinetics were determined. The half-times for secretion (t1/2) were approx. 30, 53 and 75 min for albumin, prothrombin and transferrin, respectively, whereas the minimal transit time for prothrombin was approx. 30 min, and those for albumin and transferrin 15-20 min. After injection of vitamin K-1 into warfarin-treated rats, the accumulated prothrombin precursor was gamma-carboxylated and secreted with a t1/2 of 37 min. This indicates that the gamma-carboxylation of prothrombin in rough endoplasmic reticulum cannot account for the delay in the transport of prothrombin as compared to albumin. Comparison of the incorporation of [3H]leucine and [3H]glucosamine into plasma prothrombin and transferrin suggested that transferrin is secreted randomly from an intracellular pool, whereas prothrombin is transported in a more orderly sequence. Moreover, treatment of rough microsomes with 0.05% sodium deoxycholate indicated that prothrombin is more tightly associated with the membranes of rough endoplasmic reticulum than albumin and transferrin.