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PubMed1986

Complement and neutrophil-mediated injury of perfused rat lungs.

It has previously been shown that systemic complement activation in rats leads to acute lung injury. We have now employed an ex vivo model of perfused rat lung which allows a more detailed examination of the role of circulating blood elements and pulmonary vascular pressure changes in the acute lung injury that occurs following cobra venom factor (CVF)-induced complement activation. In whole blood perfused lungs CVF infusion resulted in lung leukocyte sequestration, pulmonary vascular constriction, and acute lung injury. Lung injury was measured by quantitating the accumulation of [125I]-bovine serum albumin in lung parenchyma and alveolar lavage fluid. A histologic analysis of CVF-induced lung injury revealed the development of intravascular aggregates of platelets and accumulation of neutrophils, extensive bleb formation of pulmonary capillary interstitial endothelial cells, and interstitial and intraalveolar edema and intraalveolar hemorrhage. Experiments in lungs perfused with salt solution to which various blood elements were added showed that the development of lung injury was dependent on neutrophils and the interaction of CVF with heat-labile plasma components. The lung injury was not dependent on the presence of platelets or the pulmonary artery pressor response. Addition of catalase or erythrocytes to the lung perfusate significantly attenuated the acute lung injury. Like the development of lung injury, the pulmonary artery pressor response was dependent on the interaction of CVF with heat labile plasma components and neutrophils. The increase in PA pressure was not attenuated by the addition of catalase to the lung perfusate. These studies suggest that intravascular activation of the complement system leads to an acute microvascular injury which is dependent on neutrophils and the production of toxic oxygen metabolites. Pulmonary vascular constriction occurs independently of the associated lung injury and does not appear to be dependent on hydrogen peroxide production.

Animals↗
PubMed1999

Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis.

A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A2 (sPLA2) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA2. In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA2. On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product.

Arachidonic Acid↗
PubMed2004

Quantitative PCR analysis reveals a high incidence of large intragenic deletions in the FANCA gene in Spanish Fanconi anemia patients.

Fanconi anaemia is an autosomal recessive disease characterized by chromosome fragility, multiple congenital abnormalities, progressive bone marrow failure and a high predisposition to develop malignancies. Most of the Fanconi anaemia patients belong to complementation group FA-A due to mutations in the FANCA gene. This gene contains 43 exons along a 4.3-kb coding sequence with a very heterogeneous mutational spectrum that makes the mutation screening of FANCA a difficult task. In addition, as the FANCA gene is rich in Alu sequences, it was reported that Alu-mediated recombination led to large intragenic deletions that cannot be detected in heterozygous state by conventional PCR, SSCP analysis, or DNA sequencing. To overcome this problem, a method based on quantitative fluorescent multiplex PCR was proposed to detect intragenic deletions in FANCA involving the most frequently deleted exons (exons 5, 11, 17, 21 and 31). Here we apply the proposed method to detect intragenic deletions in 25 Spanish FA-A patients previously assigned to complementation group FA-A by FANCA cDNA retroviral transduction. A total of eight heterozygous deletions involving from one to more than 26 exons were detected. Thus, one third of the patients carried a large intragenic deletion that would have not been detected by conventional methods. These results are in agreement with previously published data and indicate that large intragenic deletions are one of the most frequent mutations leading to Fanconi anaemia. Consequently, this technology should be applied in future studies on FANCA to improve the mutation detection rate.

Alu Elements↗
PubMed1964

INHIBITION OF ANTIBODY PLAQUE FORMATION BY SENSITIZED LYMPHOID CELLS: RAPID INDICATOR OF TRANSPLANTATION IMMUNITY.

Spleen cell suspensions obtained from mice immunized with sheep erythrocytes form localized zones of hemolysis ("antibody plaques") when incubated in agar gel containing sheep red blood cells and complement. Plaque formation can be inhibited by prior incubation in vitro with spleen cell suspensions from another strain of mice previously sensitized to the first by spleen cell transplant. Suppression of plaque formation was found to be quantitatively related to the number of cells incubated and apparently reflected a homograft reaction in vitro of one spleen cell population against another. Plaque inhibition may be a useful indicator of transplantation immunity.

Animals↗
PubMed2005

Analysis of N-acylhomoserine lactones after alkaline hydrolysis and anion-exchange solid-phase extraction by capillary zone electrophoresis-mass spectrometry.

A quantitative, specific, and sensitive method for the determination of N-acylhomoserine lactones (HSLs - a group of bacterial semiochemicals) in the form of their hydrolysis products (N-acylhomoserines, HSs) is presented. Real samples were analyzed by capillary zone electrophoresis-mass spectrometry (CZE-MS) after alkaline lactonolysis and extraction by mixed-mode anion-exchange solid-phase extraction. The presented cleanup significantly speeds up the HSL extraction procedure, strongly reduces sample consumption, and is more selective compared to the commonly used liquid/liquid extraction. Completeness of the hydrolysis reaction was examined by nuclear magnetic resonance spectroscopy. This CZE-MS method complements recently published capillary separation techniques (nano liquid chromatography-MS, partial-filling micellar electrokinetic chromatography-MS, gas chromatography-MS) and provides a possibility to differentiate quantitatively between the homoserines (as naturally occurring degradation products) besides the intact homoserine lactones. The method was found to be quantitative down to a concentration of 0.05 microg/mL (limit of quantification), while the limit of detection was determined with 0.01 microg/mL - sufficient for the analysis of culture supernatants.

4-Butyrolactone↗
PubMed1983

Abnormal reticuloendothelial function in patients with active vasculitis and idiopathic membranous glomerulopathy. A study with 99mTc-labeled heat-damaged autologous red blood cells.

Reticuloendothelial function was assessed in 11 patients with systemic lupus erythematosus, 8 patients with Wegener's granulomatosus, and 20 patients with idiopathic membranous glomerulopathy by using autologous 99mTc-labeled heat-damaged red blood cells. With this method organ uptake could be measured by quantitative scintigraphy. There was no relation between the T1/2 of the blood disappearance curve and the T1/2 of the splenic uptake curve. The T1/2 of the blood disappearance curve was normal in all three patient groups. However, there was a significant shift from spleen to liver uptake in patients with active systemic lupus erythematosus, active Wegener's granulomatosus, and membranous glomerulopathy in comparison with a control group. There was no relation with age, level of circulating immune complexes, complement level, kidney function, or immunosuppressive treatment. We conclude that an increase of the liver component of reticulo-endothelial function may compensate abnormalities in splenic function. This stresses the importance of quantitative scanning to detect such abnormalities. The study provides evidence for disease related hyposplenism in patients with active systemic lupus erythematosus, active Wegener's granulomatosus, and membranous glomerulopathy.

Adult↗
PubMed1975

Viral hepatitis, type A. Identification by specific complement fixation and immune adherence tests.

Hepatitis A antibody was detected by specific immune adherence and complement-fixation tests in a study involving 473 serum specimens from 20 patients who had viral hepatitis, Type A. In all 20 patients who had no detectable immune adherence antibody (less than 1:5) before onset of hepatitis high levels (greater than or equal to 1:1024) developed one to four weeks later, occasionally reaching peak levels exceeding greater than or equal to 1:81,920 several months thereafter. Five to 10 years later the immune adherence antibody levels ranged between 1:640 and 1:20,480. In general, the complement-fixation test was not as sensitive or as specific as the immune adherence test. These findings indicate that the immune adherence test should be a valuable tool for diagnosis, for epidemiologic surveys, for identification of susceptible and immune persons, for quantitative assays of gamma globulin and for identification of hepatitis A virus in attempts to propagate the virus in cell culture.

Animals↗
PubMed1963

THE CROSS-REACTIVITY AND TRANSFER OF ANTIBODY IN TRANSPLANTATION IMMUNITY.

A close inomunochemical relationship of Forssman-type antigens in cells or in a methanol extract of guinea pig kidneys, lymph nodes, and platelets, of horse kidneys, and of sheep erythrocytes was demonstrated by complement-fixation, agglutination, and inhibition of hemolysis. The dissociation of antibody from several cross-reacting complexes and re-association with antigens of erythrocytes used for immunization was inferred from quantitative hemolytic assays. This preferential affinity of antibody for the antigen used for immunization is proposed as an immunochemical model for reactions which function in graft rejection phenomena wherein the donor and recipient tissues share cross-reacting antigens.

Agglutination↗
PubMed1992

[Significance of the complement fixation reaction in the diagnosis of the acute phase of lymphatic toxoplasmosis].

In a group of 300 sera of subjects with clinically suspect nodular toxoplasmosis the authors tested by the IgM ELISA reaction in the reverse order the importance of the complement-fixation reaction for assessment of the acute stage of the infection. In view of the revealed prolonged detection of IgM antibodies the authors used quantitative evaluation of the reaction; they consider based on a probability pattern of antibody response coefficients of 10 and above important for differentiation of a recent infection. For the suggested method of evaluation it was confirmed that low CFR titres (1:8, 1:16, 1:32) imply usually (89.5%) latent infection, titres of 1:64 chronic (42.8%) and latent infection (57.2%); in no instance onset of the disease was involved. From the titre of 1:128 onwards there is a proportional increase of the probable detection of acute infections (titre 1:1024 as much as 72.7%. However, even high titres are not unequivocal evidence of recent infection. In view of the equivocal interpretation of results of the CFR the authors recommend for assessment of the acute stage of the nodular form of toxoplasmosis examination of the suspect subject by the CFR and in case of a medium or high level assessment of IgM by the ELISA method focused on the level and dynamics of the coefficient.

Acute Disease↗
PubMed1982

Antibody activity and complement-mediated solubilization of precipitates during the immune response.

The avidity of antibodies against human serum albumin (HSA) and the complement-mediated solubilization of HSA anti-HSA precipitates has been investigated during the immune response in rats, mice, rabbits, and guinea-pigs. The avidity of the anti-HSA antibodies was quantitatively determined and expressed as the amount of HSA required to solubilize 50% of 10 ng HSA anti-HSA precipitate. In rats, from 25 to 100 days after primary immunization the avidity of the anti-HSA antibodies increased approximately 100-fold, whereas the affinity did not change significantly. The increase in avidity was accompanied by a thirty-six-fold decrease in the amount of immune precipitate that could be solubilized by complement. Booster injections of the rats did not prevent the increase in avidity (thirteen-fold) that occurred from 25 to 50 days after the first immunization; however, the subsequent eight-fold increase in avidity from 50 to 100 days could partially or completely be abolished by secondary immunizations. A close, inverse relationship between the avidity of the anti-HSA antibodies and the rate of complement-mediated solubilization was observed. In addition, an increase in avidity without a change in affinity of anti-HSA antibodies and a decrease in the rate of complement-mediated solubilization was found during the immune response against HSA not only in rats, but also in mice, rabbits, and guinea-pigs.

Animals↗
PubMed1984

Functional significance of the regulation of macrophage Ia expression.

The relationship of Ia expression and antigen-presenting function by macrophages has been evaluated. When macrophages are maintained in standard culture media, both Ia antigens and accessory cell function are lost. The reacquisition of these properties follows exposure to an Ia-inducing lymphokine, for which cDNA-derived interferon-gamma may substitute. The induction of function is related quantitatively to the level of Ia expression. Moreover, both properties reflect newly expressed Ia determinants, since treatment with anti-I-A plus complement at the beginning of culture diminishes neither the subsequent level of Ia expression nor function. Treatment with anti-Mac-1 plus complement, however, reduces function commensurate with the effectiveness of macrophage depletion. Finally, we find that fixation of macrophages after exposure to antigen does not inhibit antigen presentation, indicating that metabolic activity, while required for antigen processing, is not necessary for presentation.

Animals↗
PubMed1993

Persistent dysregulation of IgA production and IgA nephropathy in the B6C3F1 mouse following withdrawal of dietary vomitoxin (deoxynivalenol).

To assess whether vomitoxin-induced dysregulation of IgA production and IgA nephropathy are reversible, relevant immunologic parameters were compared among experimental groups of B6C3F1 mice that were fed: (1) 25 ppm vomitoxin in AIN-76A semipurified diet for 24 weeks (treatment group), (2) 25 ppm vomitoxin for 8 weeks and then control diet for 16 weeks (withdrawal group), and (3) control diet for 24 weeks (control group). Levels of serum IgA and microhematuria index in the treatment group were elevated after 4 to 8 weeks and continued to increase with further vomitoxin exposure. IgA immune complexes and mesangial IgA deposition, as quantitated by interactive laser cytometer image analysis, were also increased with toxin exposure at Weeks 8, 16, and 24, whereas IgM, IgG, and complement component C3 deposition were unaffected or depressed. Serum IgA, microhematuria index, and mesangial IgA deposition in withdrawal mice remained elevated over those of the controls at Weeks 16 and 24 but were less than those of the treatment group. Cell recovery from Peyer's patches (PP) as well as the percentages of IgA+ and CD4+ cells in PP and spleen at Weeks 16 and 24 were greater in treatment mice than in controls, but only the percentage of IgA+ cells in PP was elevated in the withdrawal mice at these the same time points. When IgA secretion by unstimulated and LPS-stimulated splenic lymphocytes was used as the measure of systemic production, it was elevated in both treatment and withdrawal mice at Weeks 16 and 24. The results indicated that experimental dysregulation of IgA production and IgA nephropathy persisted up to 4 months after a discrete period of dietary vomitoxin exposure, but that the severity of these effects did not increase in a progressive fashion.

Animals↗
PubMed2002

[Ultrasound diagnosis of the posterior tibial tendon].

Standardized sonographic delineation of the posterior tibial tendon using high-frequency ultrasonography with quantitative evaluation of the transverse section may confirm or exclude the clinical suspicion of posterior tibial tendon dysfunction and can serve as a complement to magnetic resonance imaging.

Ankle Injuries↗
PubMed2005

Genomics and proteomics: role in the management of multiple sclerosis.

Epidemiological studies and neuro-imaging have provided important insights into the natural course and prognostic factors of multiple sclerosis (MS), but our ability to predict different courses of the disease, and especially its response to treatment, is still very limited. Pharmacogenetic, pharmacogenomic and proteomic studies aim to assess gene and protein function in disease and promise to help to fill this important gap in our knowledge. Such studies may increase our understanding of disease mechanisms and responses to therapeutic compounds. Large-scale transcriptional expression profiling can be performed using gene chip microarrays; this technology allows screening for differentially expressed genes without having well-defined underlying hypotheses ("discovery-driven research"). To complement the technique, real time reverse transcription and polymerase chain reaction (RT-PCR) can be used for more targeted profiling and provides quantitative data on pre-selected genes. However, to maximise their clinical utility, expression profiling results need to be combined with well-documented clinical and imaging data. Two forthcoming studies will investigate the long-term effects of early treatment with interferon beta-1b (IFNbeta) on the course of MS. The BENEFIT (BEtaseron/Betaferon in Newly Emerging MS for Initial Treatment) study will incorporate pharmacogenetic and pharmacogenomic analyses to determine the genetic elements controlling treatment response. BEST-PGx (Betaferon/Betaseron in Early relapsing-remitting MS Surveillance Trial-Pharmacogenomics) is an exploratory 2-year study that will investigate the value of RNA expression profiling and pharmacogenetics in predicting treatment response to IFNbeta in patients with early relapsing MS. The main goal of BEST-PGx is the identification of differences in gene expression profiles of patients showing differential treatment responses. In addition, this study may reveal new information relevant to the mechanism of action of interferon treatment in MS and also to differences in the underlying pathology of the immune system. These data may help us approach the goal of a really "individualised therapy" with increased efficacy, reduced adverse drug reactions and more efficient use of health care resources.

Adjuvants, Immunologic↗
PubMed1983

An ELISA technique to detect IgG antibody to the early herpes simplex virus type 2 (HSV-2) antigen AG-4 in HSV-2 patients.

An ELISA system, based on urease activity was used for the detection and titration of IgG to the immediate early AG-4 antigen in sera from HSV-2 patients. It detected low levels of IgG to the AG-4 antigen in 32% of patients' sera known to contain complement fixing antibody to the antigen. Furthermore, the sensitivity of the ELISA system was 2- to 10-fold higher than the complement-fixation system depending on when the sera was taken from the HSV-2 patients. The system also allowed the easy detection and quantitation of AG-4 antigen production when various HSV-1 X HSV-2 intertypic recombinant viruses were used to infect BHK-21 cells.

Adult↗