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BACKGROUND: The renin-angiotensin system (RAS) protects the circulation against sudden falls in systemic blood pressure via generation of angiotensin II (AII). Previously, we demonstrated that patients with anaphylaxis involving airway angioedema and cardiovascular collapse (AACVS) had significantly increased "I" gene polymorphisms of the angiotensin-converting-enzymes (ACE). This is associated with lower serum ACE and AII levels and was not seen in anaphylaxis without collapse nor atopics and healthy controls. OBJECTIVES: To examine the angiotensinogen (AGT-M235T) and chymase gene (CMA-1 A1903G) polymorphisms in these original subjects. METHOD: 122 patients with IgE-mediated anaphylaxis, 119 healthy controls and 52 atopics had polymorphisms of the AGT gene and chymase gene examined by polymerase chain reactions and gel electrophoresis. Their previous ACE genotypes were included for the analysis. RESULTS: AGT-MM genes (associated with low AGT levels) were significantly increased in anaphylaxis (Terr's classification). When combined with ACE, anaphylaxis showed increased MM/II gene pairing (p<0.0013) consistent with lower RAS activity. For chymase, there was increased pairing of MM/AG (p<0.005) and AG/II and AG/ID (p<0.0073) for anaphylaxis consistent with lower RAS activity. A tri-allelic ensemble of the 6 commonest gene combinations for the healthy controls and anaphylaxis confirmed this difference (p=0.0001); for anaphylaxis, genes were predominately MM/AG/II or ID, while healthy controls were DD/MT/AG or GG patterns. CONCLUSION: Our gene polymorphisms show lower RAS activity for anaphylaxis especially AACVS. Animal models of anaphylaxis are focused on endothelial nitric oxide (eNO) which is shown to be the mediator of fatal shock and prevented by eNO-blockade. The interaction of AII and eNO controls the microcirculation in man. High serum AII levels reduce eNO activity, so higher RAS-activity could protect against shock. Our data shows low RAS activity in anaphylaxis especially AACVS, suggesting the influence of these genes on shock are via AII levels and its effects on eNO.
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A diagnosis of idiopathic anaphylaxis following a detailed clinical assessment remains very challenging for patients and clinicians. Risk reduction strategies such as allergen avoidance are not possible. This study investigated whether the (ISAC) allergen array with 103 allergens would add diagnostic value in patients with idiopathic anaphylaxis. We extended the specific immunoglobulin (Ig)E testing in 110 patients with a diagnosis of idiopathic anaphylaxis from five UK specialist centres using ISAC arrays. These were divided into three groups: score I identified no new allergen sensitization beyond those known by previous assessment, score II identified new sensitizations which were not thought likely to explain the anaphylaxis and score III identified new sensitizations felt to have a high likelihood of being responsible for the anaphylaxis. A proportion (50%) of score III patients underwent clinical reassessment to substantiate the link to anaphylaxis in this group. The results show that 20% of the arrays were classified as score III with a high likelihood of identifying the cause of the anaphylaxis. A wide range of major allergens were identified, the most frequent being omega-5-gliadin and shrimp, together accounting for 45% of the previously unrecognized sensitizations. The ISAC array contributed to the diagnosis in 20% of patients with idiopathic anaphylaxis. It may offer additional information where a careful allergy history and follow-on testing have not revealed the cause of the anaphylaxis.
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Alérgenos , Anafilaxia/diagnóstico , Análisis por Micromatrices/métodos , Adulto , Anciano , Alérgenos/inmunología , Anafilaxia/inmunología , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Circulating angiotensin-II protects the circulation against sudden falls in blood pressure and is generated by the enzymatic action of angiotensin converting enzyme (ACE) on angiotensin-I. The ACE genes have 2 allelic forms, "I" and "D." The "D" genotype has both highest angiotensin-II generation and serum ACE levels compared to "I". 120 patients with IgE-anaphylaxis, 119 healthy controls, and 49 atopics had serum ACE levels, ACE genotype, and renin levels determined. Plasma renin levels were identical for all groups. Serum ACE levels and genotypes were similar for healthy controls (HC) and atopics, but lower in anaphylaxis (P = 0.012), with ACE genotypes also showing increased "I" genes (P = 0.009). This effect was more pronounced in subjects manifesting airway angioedema and cardiovascular collapse (AACVS) than mild cutaneous and respiratory (CRA) symptoms. AACVS was significantly associated with the presence of "I" genes. For "ID" genotype OR is 5.6, 95% CI 1.8 to 17.4, and for "II" genotype OR is 44, 95% CI 5 to 1891 within the anaphylaxis group = 0.001. The results show a difference in the genotype frequency between control and anaphylaxis, suggesting a role for the renin angiotensin system in anaphylaxis manifesting with airway angioedema and cardiovascular collapse.
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PROBLEM: To evaluate the association of peripheral leukaemia inhibitory factor (LIF) levels on implantation and miscarriage rates after in vitro fertilization (IVF) treatment. METHODS: Prospective observational study of 120 randomly selected women who underwent IVF treatment. The concentration of LIF in serum was determined by enzyme-linked immunosorbent assay. RESULTS: There was no significant differences with regard to the systemic mean LIF concentration between the pregnant (42 patients, LIF: 11.55 pg/mL +/- 5.3 S.D.) and non-pregnant (66 patients, LIF: 13.47 pg/mL +/- 5.1 S.D.) women after IVF treatment. Likewise, for those women who have positive pregnancy after IVF treatment, the systemic mean LIF levels were not significantly different between women who have an ongoing pregnancy (34 ongoing pregnancy, LIF: 11.26 pg/mL +/- 5.2 S.D.) and those who had miscarriage (eight miscarriage, LIF: 12.78 pg/mL +/- 5.6 S.D.). CONCLUSION: The systemic levels of LIF concentration have no association with implantation rate or miscarriage rate in women undergoing IVF treatment. Measuring serum LIF concentration prior to embryo transfer in IVF treatment has no predictive value of implantation rate or miscarriage rate.
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Aborto Espontáneo/sangre , Implantación del Embrión , Fertilización In Vitro , Interleucina-6/sangre , Femenino , Humanos , Factor Inhibidor de Leucemia , Embarazo , Resultado del Embarazo , Resultado del TratamientoRESUMEN
Graft-versus-host disease (GvHD) complicates many allogeneic stem cell transplants (alloSCT), and several factors are known to be associated with the development of GvHD besides human leucocyte antigen (HLA) incompatibility. We investigated whether changes in serum levels of soluble IL-2 receptor (sIL-2Ralpha), tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), vascular endothelial growth factor (VEGF) and soluble Fas (sFas) correlated with the development of GvHD in patients undergoing SCT, and might thus be potentially of use to anticipate the development of GvHD, allowing early modification of immunosuppressive therapy.sIL2Ralpha and sFas levels were significantly raised in allograft, autograft (allo and auto) and non-graft groups compared to the normal controls (HC), but there was no statistical difference between the three patient groups. TNF-alpha was raised in the auto and allo groups and the non-graft patients compared to the HC group (median 4.37 pg/ml), but only reached significance in the allo group (median 6.02 pg/ml; p = 0.008) when this was compared with the non-graft patients. There was no significant difference in TGF-ss levels between any of the groups. The median serum VEGF levels were decreased in allo and auto patients compared to HC, (31 and 62 pg/ml versus 90 pg/ml, respectively), with a significant difference in the auto group (p = 0.007). VEGF levels were significantly lower in the auto versus the allo group (p = 0.008) and also in the auto versus the non-graft group (median 104 pg/ml; p = 0.011). When the allo group was divided into patients who developed GvHD and those who did not, serum VEGF levels were significantly higher in those with GvHD (p = 0.028).
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Citocinas/sangre , Enfermedad Injerto contra Huésped/sangre , Trasplante de Células Madre Hematopoyéticas , Biomarcadores/sangre , Anemia de Fanconi/sangre , Anemia de Fanconi/terapia , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-2 , Linfoma/sangre , Linfoma/terapia , Masculino , Valor Predictivo de las Pruebas , Receptores de Interleucina/sangre , Trasplante Autólogo , Trasplante Homólogo , Factor A de Crecimiento Endotelial Vascular/sangre , Receptor fas/sangreRESUMEN
BACKGROUND: To evaluate the association between the absolute counts of the peripheral natural killer (NK) cells (including total CD56(+) NK cells, CD56(dim) NK cells and CD56(bright) NK cells), B cells and T cells on the implantation rate and miscarriage rate after IVF treatment. METHODS: This was a prospective observation study. A total of 138 patients who underwent IVF treatment from December 2002 to July 2003 were recruited to the study. Blood samples were obtained on the day of vaginal oocyte retrieval prior to the procedure. The absolute counts of lymphocytes, NK cells, B cells and T cells were identified by flow cytometry. These absolute counts and their relationships to IVF treatment outcome and miscarriage rate were analysed. RESULTS: There were no significant differences with regard the mean values of absolute lymphocyte count, T cell count, B cell count and NK cell count (including total CD56(+) NK, CD56(dim) NK and CD56(bright) NK cells) between the pregnant and non-pregnant groups and also between the ongoing pregnancy and miscarriage groups. The cause of infertility, duration of infertility, basal FSH levels, number of previous failed IVF treatments, number of previous miscarriages and stimulation characteristics were not significantly different between the pregnant and non-pregnant groups. Previous studies have suggested that women with a history of recurrent miscarriage and those with infertility accompanied by recurrent failed IVF treatments are associated with a peripheral blood NK cell percentage >12%, therefore further analysis of peripheral CD56(+) NK cell levels <12% (group A) and >12% (group B) was performed. There was no significant difference in implantation rate (group A: 17.0%; group B: 23.2%), pregnancy rate (group A: 36.6%; group B: 47.7%) or miscarriage rate (group A: 23.3%; group B: 28.6%). CONCLUSION: There were no significant differences between simple enumerations of peripheral blood NK cells (including total CD56(+) NK, CD56(dim) NK and CD56(bright) NK cells), B cells and T cells with IVF treatment outcome and pregnancy outcome. Women who had a peripheral NK cell level >12% did not have higher number of previous pregnancy losses. Importantly their pregnancy rate was not reduced and their miscarriages were not increased compared to women who had a peripheral NK cells level <12%.
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Linfocitos B/fisiología , Fertilización In Vitro/métodos , Células Asesinas Naturales/fisiología , Linfocitos T/fisiología , Aborto Habitual/sangre , Aborto Habitual/inmunología , Adulto , Antígeno CD56 , Implantación del Embrión/inmunología , Femenino , Humanos , Valor Predictivo de las Pruebas , Embarazo , Resultado del Embarazo , Índice de Embarazo , Estudios Prospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: Our aim was to evaluate the effect of the absolute count of the activation marker (CD69), IgG Fc receptor (CD16) and inhibitor marker (CD94) expression on peripheral blood natural killer (NK) cells on implantation and miscarriage rates after IVF treatment. METHODS: Prospective observational study of 138 randomly selected women who underwent IVF treatment from December 2002 to September 2003. NK cells were identified as CD56(+) (dim + bright) and CD3(-) by flow cytometry. The absolute counts of the CD69(+), CD16(+) and CD94(+)expressing NK cells were recorded and their relation to IVF treatment outcome and miscarriage rate was analysed. RESULTS: The mean (+/-SD) absolute count of the CD56(dim)CD16(+)CD69(+) NK cells for women who had a successful ongoing pregnancy was 0.61 x 10(6)/l (+/-0.31). For those women who failed to achieve a pregnancy, the mean value of the absolute count of CD56(dim)CD16(+)D69(+) NK cells was significantly (P=0.003) higher at 1.66 x 10(6)/l (+/-0.52). The absolute count of CD56(dim)CD16(+)CD94(+) and CD56(dim)CD16(+) NK cells did not show any statistically significant differences between those women with successful and failed IVF treatment. Receiver operating characteristic (ROC) curve analysis was performed to select a CD69 threshold for further statistical analysis. The implantation rate (IR) was significantly lower (13.1%) and miscarriage rate (MR) was significantly higher (66.7%) for women with an absolute CD56(dim)CD16(+)CD69(+) NK cell count of >1.0 x 10(6)/l compared to women with count below this value (IR 28.2% and MR 16.7%). Further analysis of the absolute count of CD56(bright)CD69(+) and CD56(bright)CD94(+) NK cells did not show any significant difference between those women with successful and failed IVF treatment. CONCLUSIONS: An increase in the absolute count of activated NK cells (CD56(dim)CD16(+)CD69(+)) in the peripheral blood is associated with a reduced rate of embryo implantation in IVF treatment. Furthermore, women with high CD56(dim)CD16(+)CD69(+) peripheral blood NK cell absolute count, who are able to achieve pregnancy, have a significantly higher miscarriage rate.
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Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Antígeno CD56/análisis , Fertilización In Vitro , Infertilidad Femenina/sangre , Células Asesinas Naturales/inmunología , Resultado del Embarazo , Receptores de IgG/análisis , Aborto Espontáneo/epidemiología , Adulto , Implantación del Embrión , Femenino , Humanos , Incidencia , Infertilidad Femenina/terapia , Células Asesinas Naturales/patología , Lectinas Tipo C , Recuento de Linfocitos , EmbarazoRESUMEN
We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.
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Acetilesterasa/genética , Elementos Transponibles de ADN , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium/genética , ADN Bacteriano/química , Desoxirribonucleasa BamHI/metabolismo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Shigella flexneri/genética , Shigella flexneri/patogenicidad , Relación Estructura-ActividadRESUMEN
Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.
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Enfermedad de Crohn/microbiología , Infecciones por Mycobacterium , Mycobacterium avium subsp. paratuberculosis , Mycobacterium avium , Animales , Antibacterianos/uso terapéutico , Vacunas Bacterianas , ADN Bacteriano/análisis , Variación Genética , Humanos , Infecciones por Mycobacterium/diagnóstico , Infecciones por Mycobacterium/fisiopatología , Infecciones por Mycobacterium/prevención & control , Infecciones por Mycobacterium/transmisión , Infecciones por Mycobacterium/veterinaria , Mycobacterium avium/genética , Mycobacterium avium/aislamiento & purificación , Mycobacterium avium/patogenicidad , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/aislamiento & purificación , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Fenotipo , Reacción en Cadena de la Polimerasa , Abastecimiento de AguaRESUMEN
Trypsinogen was identified in cerebrospinal fluid (CSF), where it has not previously been reported and its activation state in experimental subarachnoid haemorrhage (SAH) in rats and in neurosurgical patients was determined. Trypsinogen activation peptide (TAP) release provided an equimolar marker for trypsinogen. Total TAP was significantly reduced to 26% of the baseline level (P<0.02) following experimental SAH in 15 rats but not in ten sham operated controls (P=0.3). TAP was also measured in patients with ruptured (n=11) and unruptured (n=9) aneurysms who underwent craniotomy to clip an aneurysm. Postoperatively there was a significant fall in TAP concentration (P<0.005) in both groups. Trypsinogen, as identified by CSF levels of TAP, is activated by SAH in rats and by craniotomy for aneurysmal clipping in patients.
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Isquemia Encefálica/líquido cefalorraquídeo , Hemorragia Subaracnoidea/líquido cefalorraquídeo , Tripsinógeno/líquido cefalorraquídeo , Animales , Apoptosis , Modelos Animales de Enfermedad , Humanos , Aneurisma Intracraneal/líquido cefalorraquídeo , Masculino , Estudios Prospectivos , Ratas , Factores de TiempoRESUMEN
Enterostatins [Val-Pro-Asp-Pro-Arg (VPDPR), Val-Pro-Gly-Pro-Arg (VPGPR), and Ala-Pro-Gly-Pro-Arg (APGPR)] are pentapeptides derived from the NH2-terminus of procolipase after tryptic cleavage and belong to the family of gut-brain peptides. Although enterostatin-like immunoreactivities exist in blood, brain, and gut, and exogenous enterostatins decrease fat appetite and insulin secretion in rats, the roles of these peptides in human obesity remain to be examined. To determine whether VPDPR and APGPR secretion is altered in obesity, serum VPDPR and APGPR levels were measured in 38 overnight-fasted subjects (body mass index, 17.9-54.7 kg/m2) before and after a meal. The mean fasting VPDPR in the serum of lean subjects was significantly lower than that in obese subjects [lean = 603 +/- 86 nmol/L (n = 17); obese, 1516 +/- 227 nmol/L (n = 21); P = 0.0023]. In addition, the rise in serum APGPR after a meal (postmeal/fasting ratio) was significantly higher in lean than in obese subjects [lean, 1.71 +/- 0.24 (n = 17); obese, 1.05 +/- 0.14 (n = 21); P = 0.0332]. The results of these studies show hyperenterostatinemia in obesity and a diminution in enterostatin secretion after satiety.
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Colipasas/sangre , Obesidad/sangre , Premenopausia/sangre , Precursores de Proteínas/sangre , Adulto , Ingestión de Alimentos/fisiología , Precursores Enzimáticos , Ayuno , Femenino , Humanos , Oligopéptidos/sangreRESUMEN
Enterostatins belong to a family of peptides (e.g., Val-Pro-Asp-Pro-Arg, VPDPR; Ala-Pro-Gly-Pro-Arg, APGPR; and Val-Pro-Gly-Pro-Arg, VPGPR) derived from the tryptic cleavage of amino-terminal pentapeptide from procolipase. Pharmacologic studies have suggested a role for these peptides in appetite regulation and insulin secretion. Studies into the distribution of enterostatins or the role of endogenous peptides have not been possible until now due to the lack of a suitable method for assay. Using two polyclonal antibodies raised against VPDPR and APGPR and different chromatographic methods, we have examined the nature and distribution of enterostatin-like immunoreactivity in human cerebrospinal fluid. The results reported here show for the first time the presence of enterostatin-like immunoreactivity in the human cerebrospinal fluid. Further characterization of cerebrospinal fluid enterostatin-like immunoreactivity revealed that it is not due to APGPR, VPGPR, or VPDPR but to another peptide similar to VPDPR.
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Colipasas/líquido cefalorraquídeo , Precursores de Proteínas/líquido cefalorraquídeo , Adulto , Factores de Edad , Anciano , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Precursores Enzimáticos , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oligopéptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/líquido cefalorraquídeoRESUMEN
This long-term prospective study of patients with newly diagnosed RA assesses the relative value of certain clinical and laboratory measures at first consultation in order to determine factors that help to discriminate between patients likely to go into early remission and those with relapsing/remitting or persistent disease. Validation was sought in a similar group from a separate but comparable prospective study. Measures of clinical activity such as joint score, early morning stiffness (EMS) and acute phase (ESR) improved over 4 yr in both groups, whereas agalactosyl IgG [Gal(o)] levels increased. Using discriminant functional analysis in the first cohort to identify features at entry which would predict outcome at 4 yr, a combination of Gal(o), grip strength, age at onset and gender predicted the course of RA correctly in 95% of the patients. This combination was confirmed in the second cohort, although with reduced power (78% correct). Thus, we show that Gal(o) does not reflect activity of the disease like the ESR, but has greater potential as a prognostic index early in the course of disease.
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Artritis Reumatoide/sangre , Inmunoglobulina G/sangre , Sedimentación Sanguínea , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Patients with rheumatoid arthritis (RA) have a higher proportion of agalactosyl IgG than healthy individuals. Glycosylation status was examined in 26 RA patients who fasted for 7-10 days and afterwards followed a vegetarian diet for 3.5 months. The decrease in the proportion of agalactosyl IgG correlated significantly with the clinical improvement after the fasting period, but not after the vegetarian diet period. Although the glycosylation status of IgG may have played a role in the improvement of disease during the fasting period, it did not seem to be associated with, and therefore responsible for, the clinical improvement observed after the vegetarian diet.
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Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Ayuno/fisiología , Inmunoglobulina G/metabolismo , Adulto , Dieta , Dieta Vegetariana , Femenino , Glicosilación , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Using an assay for measurement of released type 1-prophospholipase A2 (type 1-proPLA2) propeptides (PROP assay), we have shown that human granulocytes, but not lymphocytes or macrophages, abundantly express this 'pancreatic' type 1-proPLA2 zymogen. Stimulation with tumour necrosis factor-alpha (TNF-alpha) and other cytokines results in the immediate release from granulocytes of a mixture of free propeptides and type 1-proPLA2 precursor. We also found that granulocytes contain an approximately 29 kDa trypsin-like endogenous type 1-proPLA2 activator. PROP assay and TAP (trypsinogen activation peptide) assay of plasma samples accurately predicts the segregation of acute pancreatitis into three clearly defined categories of severity--mild, intermediate and severe--at the time of first hospital admission and over the next few hours of observation. Mild and intermediate pancreatitis are associated with a degree of granulocyte stimulation limited to the release of the unactivated type 1-proPLA2 precursor. Progression to severe disease is accompanied by the activation of granulocyte type 1-proPLA2, apparently carried to completion. This identifies the approximately 29 kDa endogenous activator of type 1-proPLA2 in granulocytes as a critical mediator at a threshold stage in acute pancreatitis, which marks the transition from uncomplicated pancreatitis to the potentially lethal disease. Specific inhibitors of this key regulatory enzyme modelled on the P3-P1 domain of the type 1-proPLA2 activation peptide would seem to be promising candidates for a new class of chemotherapeutic agents.
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Precursores Enzimáticos , Granulocitos/enzimología , Pancreatitis/fisiopatología , Fosfolipasas A/metabolismo , Precursores de Proteínas/metabolismo , Activación Enzimática , Humanos , Pancreatitis/sangre , Fosfolipasas A2 , Señales de Clasificación de ProteínaRESUMEN
To study the early pathogenesis of acute edematous pancreatitis in dogs, we examined the relationship of pancreatic hyperstimulation with cholecystokinin-8 (10 micrograms/kg/hr intravenously for 6 hr) to alterations in circulating pancreatic enzymes and pancreatic morphology with special reference to trypsinogen activation. Cholecystokinin-8 infusion was associated with increases in plasma amylase, lipase, trypsin-like immunoreactivity, and plasma and urine trypsinogen activation peptide. Pancreatic parenchymal swelling and interlobular and subcapsular fluid accumulations were detected ultrasonographically within 2 hr of cholecystokinin-8. Circulating trypsin-like immunoreactivity and trypsinogen activation peptide in urine reached a peak at 2 and 4 hr, respectively, then declined despite progressive increases in circulating amylase and lipase and intrapancreatic fluid. No significant changes were observed in dogs receiving a saline infusion. This study illustrates that cholecystokinin-8 induces edematous pancreatitis in dogs that is associated with a short-lived burst of trypsinogen activation.
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Edema/inducido químicamente , Pancreatitis/inducido químicamente , Sincalida/toxicidad , Tripsinógeno/efectos de los fármacos , Enfermedad Aguda , Animales , Perros , Edema/diagnóstico por imagen , Edema/metabolismo , Edema/patología , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Oligopéptidos/análisis , Oligopéptidos/efectos de los fármacos , Oligopéptidos/metabolismo , Páncreas/diagnóstico por imagen , Páncreas/enzimología , Páncreas/patología , Pancreatitis/diagnóstico por imagen , Pancreatitis/metabolismo , Pancreatitis/patología , Factores de Tiempo , Tripsinógeno/análisis , Tripsinógeno/metabolismo , UltrasonografíaRESUMEN
OBJECTIVE: To establish a ELISA assay to measure release of type 1-phospholipase A2 propeptide from activated granulocytes. Human type 1-prophospholipase A2 (1-proPLA2) is biosynthesized and stored as inactive zymogen. Activation involves tryptic-like cleavage at the N-terminus, with equimolar release of the heptapeptide DSGISPR. METHODS: Using antibodies directed to the carboxyterminus of synthetic DSGISPR we developed a sensitive solid-phase ELISA specific for the released propeptide that accurately reports the activation of 1-proPLA2. The presence of the 1-proPLA2 precursor itself can be determined by trypsinization of the sample and subsequent assay for free DSGISPR. RESULTS: Using this ELISA, we demonstrated the presence of immunoreactive DSGISPR and its 14 kDa 1-proPLA2-like precursor in human granulocytes, but their absence in human macrophages and lymphocytes. Stimulation of cultured granulocytes with 1 pM of TNF alpha or GM-CSF caused rapid release of DSGISPR and precursor into the surrounding medium. The immunoreactive signal coeluted with standard synthetic DSGISPR on G50 Sephadex chromatography. CONCLUSION: Release of DSGISPR immunoreactivity appears to be a specific consequence of granulocyte activation of potential relevance to the clinical pathophysiology of conditions like acute lung injury.
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Precursores Enzimáticos , Ensayo de Inmunoadsorción Enzimática/métodos , Granulocitos/enzimología , Fosfolipasas A/inmunología , Fosfolipasas A/farmacocinética , Precursores de Proteínas/inmunología , Precursores de Proteínas/farmacocinética , Secuencia de Aminoácidos , Citocinas/metabolismo , Granulocitos/química , Granulocitos/fisiología , Humanos , Linfocitos/química , Linfocitos/enzimología , Linfocitos/metabolismo , Datos de Secuencia Molecular , Monocitos/química , Monocitos/enzimología , Monocitos/metabolismo , Fosfolipasas A/sangre , Fosfolipasas A2 , Precursores de Proteínas/sangre , Tripsina/químicaRESUMEN
Neutrophil sequestration and activation in the pulmonary vasculature and interstitium are important in acute lung injury. Phospholipase A2 plays an important part in the production of potent inflammatory mediators in this syndrome. We used our ELISA for type 1 prophospholipase A2 activation peptides, which have the aminoacid sequence Asp-Ser-Gly-Ile-Ser-Pro-Arg (DSGISPR), to show that DSGISPR concentrations in plasma and urine are a sensitive and specific marker of acute lung injury in patients admitted to intensive care. The detection of DSGISPR in the plasma of 11 of 50 unselected patients had a sensitivity of 100% and a specificity of 93% for the presence or future development of acute lung injury.
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Precursores Enzimáticos , Enfermedades Pulmonares/enzimología , Fosfolipasas A/sangre , Precursores de Proteínas/sangre , Secuencia de Aminoácidos , Biomarcadores , Ensayo de Inmunoadsorción Enzimática , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Enfermedades Pulmonares/inmunología , Datos de Secuencia Molecular , Neutrófilos/inmunología , Fosfolipasas A/metabolismo , Fosfolipasas A/orina , Fosfolipasas A2 , Precursores de Proteínas/metabolismo , Precursores de Proteínas/orina , Sensibilidad y EspecificidadRESUMEN
MRL-lpr/lpr strain mice have defectively glycosylated IgG. This may be related to the rheumatoid arthritis (RA)-like disease that occurs in these mice, because a similar glycosylation defect is seen in human subjects with RA. Whilst it is known that this defect is associated with reduced activity of the beta-1,4-galactosyltransferase (beta-1,4-GalTase) enzyme, the cause of this reduced activity is at present unknown. We have therefore examined the molecular genetics of beta-1,4-GalTase in MRL-lpr/lpr mice. Using 10 different restriction endonucleases we found no evidence for a polymorphic variant of the gene in glycosylation-defective mice. However, the level of mRNA for beta-1,4-GalTase was lowest in the MRL-lpr/lpr mice, the strain with the most poorly galactosylated IgG of the four strains examined. Thus, the reduced level of IgG oligosaccharide galactosylation found in MRL-lpr/lpr strain mice appears to be related to either an altered transcriptional level of, or altered mRNA stability for, beta-1,4-GalTase in lymphocytes from these mice.