RESUMO
Low-grade proteinuria and systolic hypertension (SHT) are risk factors for allograft failure. Both are dynamic variables and their relationship is not independent. We have simultaneously analyzed the effects of proteinuria and SHT on graft outcomes in 805 adult Kidney Transplant Recipients and impact of their changes over time. Proteinuria and systolic blood pressure (SBP) were recorded for years 1 and 3 posttransplantation. Subjects with proteinuria >1 g/day were excluded. Patients were divided into groups based on proteinuria (Absent(A) <150 mg/day or low-grade(P)150 mg-1 g/day) and blood pressure (Normotensive-SBP <140 mmHg or hypertensive-SBP ≥ 140 mmHg). Graft survival was assessed in all four groups over 10 years by multivariate analysis. At the three annual time points (Year 1, 2 and 3) hypertensive patients with proteinuria had the worst graft survival. Patients with persistent proteinuria between years 1-2 and 2-3 had the poorest graft survival with an improvement if proteinuria regressed (P-A), especially in the Hypertensive group. The impact of proteinuria was highest in persistently hypertensive patients between years 1-3. Thus both proteinuria and SHT were associated with poor graft survival and the combination of the two led to the worst outcomes. Importantly, SHT was associated with significantly worse outcomes in patients with proteinuria. Patient cohort with SHT and low-grade proteinuria represent a selective group that might benefit from intervention.
Assuntos
Transplante de Rim , Proteinúria/fisiopatologia , Pressão Sanguínea , Sobrevivência de Enxerto , Humanos , Estudos Longitudinais , Estudos RetrospectivosRESUMO
Several studies have analyzed the phenotype of repopulated T-lymphocytes following alemtuzumab induction; however there has been less scrutiny of the reconstituted B-cell compartment. In the context of a randomized controlled trial (RCT) comparing alemtuzumab induction with tacrolimus monotherapy against basiliximab induction with tacrolimus and mycophenolate mofetil (MMF) therapy in renal transplantation, we analyzed the peripheral B- and T-lymphocyte phenotypes of patients at a mean of 25 +/- 2 months after transplantation. We examined the relationship between peripheral lymphocyte phenotype and graft function. Patients who received alemtuzumab had significantly higher numbers of B cells including naïve, transitional and regulatory subsets. In contrast, the CD4(+) T-cell compartment was dominated by a memory cell phenotype. Following either basiliximab or alemtuzumab induction patients with lower numbers of B cells or B subsets had significantly worse graft function. For alemtuzumab there was also a correlation between these subsets the stability of graft function and the presence of HLA-specific antibodies. These results demonstrate that a significant expansion of regulatory type B cells is associated with superior graft function and that this pattern is more common after alemtuzumab induction. This phenomenon requires further prospective study to see whether this phenotype could be used to customize immunotherapy.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Imunossupressores/uso terapêutico , Transplante de Rim , Linfócitos/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Alemtuzumab , Antineoplásicos/uso terapêutico , Basiliximab , Citometria de Fluxo , Humanos , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/uso terapêutico , Tacrolimo/uso terapêuticoRESUMO
The multichain immune recognition receptors (MIRRs), including the T cell and B cell antigen receptors and the high affinity receptor for IgE, play an important role in immune cell signaling. The MIRRs have no inherent kinase activity, but rather associate with members of the Src-family kinases to initiate signaling. Although a great deal is understood about the biochemical cascades triggered by MIRRs, the mechanism by which signaling is initiated was not known. The evidence now indicates that the Src-family kinases are concentrated in cholesterol- and sphingolipid-rich membrane microdomains, termed lipid rafts, that exclude the MIRRs. Upon ligand-induced crosslinking the MIRRs translocate into rafts where they are phosphorylated. The MIRRs subsequently form highly ordered, polarized structures termed immunological synapses that provide for prolonged signaling. An understanding of the biochemical composition of rafts and synapses and the mechanisms by which these form should lend insight into the regulation of immune cell activation.
Assuntos
Microdomínios da Membrana/fisiologia , Receptores Imunológicos/fisiologia , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Antígenos CD/imunologia , Antígenos CD19/imunologia , Linfócitos B/química , Linfócitos B/imunologia , Toxinas Bacterianas/metabolismo , Antígenos CD28/imunologia , Antígeno CD48 , Diferenciação Celular , Colesterol/análise , Glicoesfingolipídeos/análise , Herpesvirus Humano 4/fisiologia , Humanos , Capeamento Imunológico , Ligantes , Ativação Linfocitária , Substâncias Macromoleculares , Lipídeos de Membrana/análise , Microdomínios da Membrana/química , Microdomínios da Membrana/enzimologia , Fosforilação , Proteína Quinase C/fisiologia , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , Agregação de Receptores , Receptores de Complemento 3d/imunologia , Receptores Imunológicos/química , Esfingolipídeos/análise , Linfócitos T/química , Linfócitos T/imunologia , Proteínas da Matriz Viral/fisiologia , Quinases da Família src/fisiologiaRESUMO
A series of compounds were tested as inhibitors of receptor-binding and uptake of fluorescein-derivatized antigens in primary peritoneal and J774 murine macrophage. Results were analysed with regard to the inhibitory potency of the tested ligands and revealed that the putative cell surface receptor preferentially recognized and bound aromatic structures containing phenyl rings. L-phenylalanine was found to be the most potent inhibitor among the ligands tested. Significant inhibition of FITC10BSA binding to macrophage was observed even at 10(-9) M concentration of specific monovalent ligands tested indicating that these ligands were bound by the putative macrophage receptor with high apparent affinity. Structural comparisons of the various inhibitors employed, demonstrated that accessible, unconjugated phenyl rings were bound by the putative receptor with high apparent affinity whereas non-phenyl derivatives were bound with either low apparent affinity or via non-specific interactions. Therefore, the fluorescein hapten appeared to utilize a receptor with specificity for an essential aromatic amino acid for gaining entry into the endocytic pathway of murine macrophage. Finally, the binding of the hapten was enhanced when polyvalent fluorescein-derivatized antigens were used as a result of receptor crosslinkage on the cell surface.
Assuntos
Endocitose/imunologia , Fluoresceína , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Receptores de Superfície Celular/imunologia , Aminoácidos , Animais , Bovinos , Linhagem Celular , Haptenos/imunologia , Ligantes , CamundongosRESUMO
Fluorescein-derivatized bovine serum albumin (FITC-BSA) was used as an exogenous antigen and fluorescent probe to measure the kinetics of antigen uptake into the endocytic pathway of murine macrophage, J774, using flow cytometry. Results revealed dependency of the rate of antigen uptake on epitope density (moles FITC/mole BSA) implicating a role for FITC in the endocytosis of the derivatized antigen. In addition, inhibition of clathrin-coated pit formation in macrophage resulted in significantly reduced uptake of differentially labeled FITC BSA probes indicating receptor-mediated endocytosis via clathrin-coated pits. Fluoresceinamine (I) was found to inhibit the endocytic uptake of FITC-BSA at 10(-6) M. Determination of fractional receptor occupancies in macrophage upon binding different FITC BSA probes and calculation of the corresponding association rates (k(on)) for these binding events yielded values of 4.2+/-0.2 x 10(6)/M/min for FITC5BSA and 1.9+/-0.1 x 10(7)/M/min for FITC22BSA, respectively, at 37 degrees C. The five-fold difference in the rates of binding and endocytosis between the two probes was discussed on the basis of receptor cross-linking by a multivalent ligand (FITC22BSA), in contrast to monovalent ligand binding, on the cell surface that would lead to more rapid and efficient internalization of the FITC22BSA antigen.
Assuntos
Endocitose/imunologia , Fluoresceína-5-Isotiocianato/análogos & derivados , Haptenos/imunologia , Macrófagos/metabolismo , Receptores Imunológicos/fisiologia , Soroalbumina Bovina/metabolismo , Animais , Ligação Competitiva/imunologia , Linhagem Celular , Fenômenos Químicos , Físico-Química , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Haptenos/química , Cinética , Lisina/metabolismo , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Potássio/metabolismo , Soroalbumina Bovina/químicaRESUMO
A 48 year old patient with resistant coeliac disease developed prolonged unexplained pyrexia after surgery for small bowel volvulus. Despite extensive investigations and intensive antibiotic therapy, he deteriorated and died eight weeks postoperatively and significant isolated pulmonary valve endocarditis was discovered at autopsy. This diagnosis should be considered in all critically ill patients with unexplained pyrexia even in the absence of clinical features of endocarditis and transoesophageal echocardiography performed to exclude or confirm this lesion.
Assuntos
Endocardite Bacteriana/epidemiologia , Valva Pulmonar , Infecções Estafilocócicas/epidemiologia , Endocardite Bacteriana/complicações , Endocardite Bacteriana/diagnóstico , Febre de Causa Desconhecida/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Infecções Estafilocócicas/complicações , Infecções Estafilocócicas/diagnóstico , Fatores de TempoRESUMO
INTRODUCTION: Bisphosphonate (Bp)-ibandronate is a pharmacological agent, exhibits antiosteoclastic or antiresorptive activity and used to treat osteolytic or osteopenic disorders. BP-ibandronate may also interfere during orthodontic tooth movement. The aim of this study was to examine the influence of locally administered Bp-ibandronate on experimental tooth movement in rabbits. MATERIALS AND METHODS: Twenty rabbits were divided into two groups- "10" served as Group-1 (control) and other "10" as Group-2 (experimental). Both groups received nickel-titanium closed coil springs with 100 g force between mandibular molar and incisors. Group-1 animals received 1 ml normal saline and Group-2 animals received ibandronate solution (0.3 mg/kg body weight) locally, mesial to the mandibular molar on the 1(st), 7(th), and 14(th) day of the experiment. A total of "40" lateral cephalograms were taken from both groups on the 1(st) and 21(st) day using a digital X-ray unit (Siemens X-ray systems, 300 mA Pleomophos analog, 2008, Germany). Individually, each animal's radiograph was traced manually and superimposed. The molar tooth movement was measured with the help of a standard metric scale. RESULTS: The Student's t-test has been done to compare the mean values of Group-1 (4.650 ± 0.363) and Group-2 (2.030 ± 0.291) and the difference was statistically significant (P < 0.001). CONCLUSION: The retarded molar tooth movement was noticed in local drug administered rabbits, which could be beneficial in orthodontics to control the undesired tooth movement.
RESUMO
BACKGROUND: The change in the demographics and the presence of multiple risk factors and co-morbidities in UK patients starting dialysis may lead to poor survival on dialysis. Many of these risk factors are present in the pre-dialysis period allowing a potential window of opportunity to intervene with risk modification measures. AIM: To examine various potential factors that may predict early and overall mortality. DESIGN AND METHODS: We carried out an observational prospective study of a cohort of incident patients starting dialysis in a UK centre. Univariate analysis of factors and co-morbidities potentially affecting survival on dialysis were analysed to potential predictors. Factors affecting 1 year mortality were analysed using the t-test, the Mann-Whitney U-test or the chi-square test as appropriate. Mortality over the 5-year follow-up period was analysed using the Kaplan-Meier method. RESULTS: Ninety-four patients [predominantly Caucasian (98%)], of mean age 63 years (15.6) (56% > 65 years) with a slight male preponderance were studied. Vascular disease (39%) and sepsis (33%) accounted for most of the deaths and a significant proportion of mortality was seen in the first year (56%). Patients with early mortality were older (68 vs. 61 years, P = 0.05) with lower haemoglobin (8.4 vs. 9.4 g/dl, P = 0.01) at the start of dialysis, commenced dialysis with a lower eGFR (5.4 vs. 6.5 ml/min/1.73 m(2), P = 0.06) and had more peripheral vascular disease (PVD) (39% mortality in patients with PVD vs. 18.5% in those without PVD, P = 0.04). Diabetes mellitus, high calcium phosphate product, older age and presence of vascular co-morbidities including ischaemic heart disease and peripheral vascular disease were associated with overall mortality over the 5-year follow-up period. SUMMARY: In this study, elevated calcium phosphate product and diabetes mellitus in addition to the presence of vascular disease were associated with poor survival. Patients with low haemoglobin and lower first pre-dialysis eGFR suffered higher early mortality. These potentially modifiable factors that could be identified in the pre-dialysis stage provide a valuable opportunity for intervention.
Assuntos
Diálise Renal/mortalidade , Fatores Etários , Idoso , Análise de Variância , Fosfatos de Cálcio/sangue , Diabetes Mellitus/epidemiologia , Inglaterra/epidemiologia , Feminino , Hemoglobinas/análise , Humanos , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Análise de Sobrevida , Doenças Vasculares/epidemiologiaRESUMO
The B cell antigen receptor (BCR) is a member of an important family of multichain immune recognition receptors, which are complexes composed of ligand-binding domains associated with signal-transduction complexes. The signaling components of these receptors have no inherent kinase activity but become tyrosine phosphorylated in their cytoplasmic domains by Src-family kinases upon oligomerization, thus initiating signaling cascades. The BCR is unique in this family in that, in addition to its signaling function, it also serves to deliver antigen to intracellular compartments where the antigen is processed and presented bound to major histocompatibility complex (MHC) class II molecules. Recent evidence indicates that both the signaling and antigen-trafficking functions of the BCR are regulated by cholesterol- and sphingolipid-rich plasma membrane microdomains termed rafts. Indeed, upon oligomerization, the BCR translocates into rafts that concentrate the Src-family kinase Lyn and is subsequently internalized directly from the rafts. Thus, translocation into rafts allows the association of the oligomerized BCR with Lyn and the initiation of both signaling and trafficking. Significantly, the access of the BCR to rafts appears to be controlled by a variety of B lymphocyte co-receptors, as well as factors including the developmental state of the B cell and viral infection. Thus, the translocation of the immune receptors into signaling-competent microdomains may represent a novel mechanism to initiate and regulate immune-cell activation.
Assuntos
Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/imunologia , Animais , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Modelos Imunológicos , Transporte ProteicoRESUMO
The CD19/CD21 complex is an essential B cell coreceptor that functions synergistically to enhance signaling through the B cell Ag receptor in response to T cell-dependent, complement-tagged Ags. In this study, we use a recombinant protein containing three tandemly arranged copies of C3d and the Ag hen egg lysozyme, shown to be a highly effective immunogen in vivo, to evaluate the role of the CD19/CD21 complex in Ag processing in B cells. Evidence is provided that coengagement of the CD19/CD21 complex results in more rapid and efficient production of antigenic peptide/class II complexes as compared with B cell Ag receptor-mediated processing alone. The CD19/CD21 complex does not itself target complement-tagged Ags for processing, but rather appears to influence B cell Ag processing through its signaling function. The ability of the CD19/CD21 complex to augment processing may be an important element of the mechanism by which the CD19/CD21 complex functions to promote B cell responses to T cell-dependent complement-tagged Ags in vivo.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD19/fisiologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Complemento C3d/metabolismo , Receptores de Complemento 3d/fisiologia , Adjuvantes Imunológicos/fisiologia , Animais , Feminino , Antígenos de Histocompatibilidade Classe II/metabolismo , Ligantes , Substâncias Macromoleculares , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Muramidase/metabolismo , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosforilcolina/metabolismo , Pinocitose/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Células Tumorais CultivadasRESUMO
Binding properties and requirements for internalization of hapten-protein antigens, such as fluorescein-polyderivatized bovine serum albumin (FITC-BSA) and poly-D-lysine (FITC-PDL), by murine macrophage was consistent with surface receptor recognition of fluorescyl moieties (Cherukuri et al., Mol. Immunol. 34, 21-32, 1997; Cherukuri et al., Cytometry 31, 110-124, 1998). Ligand binding properties of the putative macrophage receptor pointed toward specificity for various aromatic moieties including phenylalanine, phenyloxazolone and fluorescein (Cherukuri and Voss, Mol. Immunol. 35, 115-125, 1998). Purification of the hapten-recognizing receptor from J774 macrophage cells involved subcellular fractionation of plasma membrane fractions, and affinity chromatography of solubilized membranes employing a phenyl-Sepharose adsorbent with subsequent specific elution of receptor using fluorescein ligand. The final product was a protein with a molecular mass of approximately 180 kDa. Characterization of the purified receptor involved absorption and fluorescence spectroscopy, circular dichroism, fluorescence quenching analyses, various ligand binding assays and an immunological analysis. Spectroscopic analyses revealed that the receptor possessed aromatic amino acids while circular dichroism suggested significant alpha-helical secondary structure. Binding specificity of the purified receptor was confirmed in a spectrofluorometric assay where the fluorescence of fluorescein ligand was quenched approximately 97%. Finally, specific binding activity of the receptor with FITC-BSA was demonstrated in Western blot analysis under native conditions. Receptor purity was confirmed in amino acid sequencing analysis when the amino-terminal residue was found to be totally blocked. Results are discussed in terms of the possible identity of the isolated macrophage receptor and its biological-immunological role.
Assuntos
Fluoresceína/metabolismo , Macrófagos/química , Oxazolona/análogos & derivados , Animais , Western Blotting , Bovinos , Linhagem Celular , Dicroísmo Circular , Feminino , Citometria de Fluxo , Fluorometria , Haptenos/química , Haptenos/imunologia , Haptenos/metabolismo , Imunoglobulina G/imunologia , Ligantes , Camundongos , Oxazolona/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Coelhos , Frações Subcelulares/químicaRESUMO
The CD19/CD21 complex functions to significantly enhance B cell antigen receptor (BCR) signaling in response to complement-tagged antigens. Recent studies showed that following antigen binding the BCR translocates into plasma membrane lipid rafts that serve as platforms for BCR signaling. Here, we show that the binding of complement-tagged antigens stimulates the translocation of both the BCR and the CD19/CD21 complex into lipid rafts, resulting in prolonged residency in and signaling from the rafts, as compared to BCR cross-linking alone. When coligated to the BCR, the CD19/CD21 complex retards the internalization and degradation of the BCR. The colocalization and stabilization of the BCR and the CD19/CD21 complex in plasma membrane lipid rafts represents a novel mechanism by which a coreceptor enhances BCR signaling.
Assuntos
Antígenos CD19/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Complemento 3d/metabolismo , Animais , Antígenos/metabolismo , Antígenos CD19/química , Transporte Biológico Ativo , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Reagentes de Ligações Cruzadas , Feminino , Hibridomas/imunologia , Ligantes , Substâncias Macromoleculares , Masculino , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Muramidase/imunologia , Ratos , Receptores de Complemento 3d/química , Transdução de SinaisRESUMO
A series of fluorescein derivatized poly-D-lysine (FITC-PDL) probes were used to elucidate the role of fluorescein in receptor binding of fluorescein-conjugated macromolecules to J774 murine macrophages. Poly-D-lysine served to eliminate receptor recognition of the carrier due to the biologically inert nature of the D-isomer. This concept enabled the focused investigation of the role played by fluorescein in receptor recognition, binding and internalization. Results revealed dependency of cellular uptake on polymer concentration, hapten density and accessibility. The results differed from those previously obtained with FITC-BSA in that saturating fluorescein densities on the poly-D-lysine polymer resulted in diminished rates of uptake by macrophages. Receptor-mediated endocytosis via clathrin-coated pits was concluded based on results that showed inhibition of FITC-PDL uptake by intracellular K+ depletion but not by the macropinocytosis inhibitor, amiloride. Further, FITC-PDL was found to inhibit the endocytic uptake of FITC-BSA suggesting competition between the two probes at the level of a macrophage receptor. Association rates (kon) for binding to the macrophage surface were measured for the various FITC-PDL probes based on fractional receptor occupancies. Results are discussed on the basis of receptor recognition of fluorescein in J774 macrophages and the requirements for this recognition which include appropriate spacing and accessibility of the hapten moieties to facilitate receptor crosslinking.
Assuntos
Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Haptenos/metabolismo , Macrófagos/citologia , Receptores de Superfície Celular/metabolismo , Amilorida/farmacologia , Animais , Ligação Competitiva , Linhagem Celular , Endocitose/fisiologia , Citometria de Fluxo/métodos , Fluoresceína-5-Isotiocianato/análogos & derivados , Cinética , Ligantes , Camundongos , Microscopia de Fluorescência , Pinocitose/efeitos dos fármacos , Potássio/fisiologia , Soroalbumina BovinaRESUMO
A 48-year-old man accidently swallowed the ring pull from a soft drink can. He complained of pain in his chest. Chest radiographs were normal. A metal detector emitted a strong response when passed across the front of his chest. Oesophagoscopy was carried out and the ring pull was successfully removed. We recommend the wider use of metal detectors by accident and emergency (A&E) department staff particularly when dealing with patients who have ingested metals of low radiodensity.
Assuntos
Alumínio , Esôfago , Corpos Estranhos/diagnóstico , Serviço Hospitalar de Emergência , Esofagoscopia , Corpos Estranhos/diagnóstico por imagem , Corpos Estranhos/terapia , Humanos , Masculino , Pessoa de Meia-Idade , RadiografiaRESUMO
The initiation of antibody responses to foreign antigens requires that B cells receive and integrate a variety of signals through an array of cell surface receptors including the B cell antigen receptor (BCR) as well as a number of essential coreceptors. Recent evidence indicates that cholesterol-rich plasma membrane microdomains, referred to here as lipid rafts, serve as platforms for BCR signaling and trafficking in B cells. The existence of rafts suggests a previously unappreciated level of organization at the B cell surface that may explain, at least in part, how BCR signaling is coordinated. Here the current evidence that lipid rafts play a key role in B cell responses is reviewed.
Assuntos
Linfócitos B/imunologia , Microdomínios da Membrana/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Antígenos/imunologia , Antígenos CD19/imunologia , Linfócitos B/citologia , Transporte Biológico , Diferenciação Celular , Herpesvirus Humano 4/imunologia , Humanos , Receptores de Complemento 3d/imunologiaRESUMO
BACKGROUND: Nitric oxide (NO) production is increased in inflammatory bowel disease (IBD), and measurement of NO metabolites may be useful for monitoring disease activity. AIMS AND OBJECTIVES: To characterise urinary nitrite levels, a stable metabolite of NO, in IBD and to evaluate its potential as a marker of disease activity. METHODS: Twelve-hour urinary nitrites were measured by the microplate assay method in 46 patients with IBD (active; n = 32). Urinary samples from 16 healthy individuals served as controls. RESULTS: Increased levels of urinary nitrites were found in patients with active IBD compared with those with inactive IBD. Twenty-eight out of 32 patients (87.5%) with active IBD had detectable levels of nitrite in their urine as compared with 2/14 (14.3%) patients with inactive IBD. None of the 16 healthy controls had detectable urinary nitrite. Twelve-hour urinary nitrite in active compared with inactive IBD: 5 0.7 versus 0.1+/-0.04 micromol (P < 0.05). There was good correlation between urinary nitrite and some markers of disease activity in IBD such as C-reactive protein and microalbuminuria but not with erythrocyte sedimentation rate. CONCLUSIONS: Increased levels of nitrite were detected in urine of patients with active IBD, consistent with increased NO synthesis. This simple assay may be exploited as a potential marker of disease activity in IBD.
Assuntos
Doenças Inflamatórias Intestinais/urina , Nitritos/urina , Albuminúria , Proteína C-Reativa/análise , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/urina , Doença de Crohn/diagnóstico , Doença de Crohn/urina , Humanos , Doenças Inflamatórias Intestinais/diagnósticoRESUMO
To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.