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New insights into complement: a mediator of injury and marker of disease activity in systemic lupus erythematosus.

Studies performed during the past several decades have demonstrated a role for the complement system in both the etiology and pathogenesis of systemic lupus erythematosus (SLE). However the specifically defective molecular and cellular pathways responsible for the disease and its complications have generally not been identified. In this report, we describe two recent advances in complement pathobiology that highlight future directions for promising investigation toward enhancing our capacity to diagnose SLE, to monitor activity of the disease, and to identify molecular and cellular defects in SLE that can be targeted by therapeutic inhibitors of complement activation. In the first example, we describe recently developed assays to detect erythrocyte C4d and complement receptor 1 for diagnosis and monitoring of disease activity in SLE. In the second example, we describe a recently discovered role for complement in mediating fetal loss in antiphospholipid syndrome and discuss the potential for this observation to facilitate identification and development of complement based biomarkers to predict poor fetal outcome in pregnant patients with SLE. These two examples are meant to underscore the importance of complement in the etiology and pathogenesis of SLE and its complications, and to stress the need for further investigation focused on the link between the complement system and SLE.

Biomarkers↗

Hypoxia-induced mitochondrial and nuclear DNA damage in the rat brain.

In humans, cerebral hypoxia is a common component of severe brain insults, including trauma, stroke, and perinatal asphyxia. Oxidative stress and free radicals incidental to cerebral hypoxia are implicated in damaging macromolecules, leading to collapse of cellular homeostasis and cell death. Neuronal DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Here, we measured hypoxia-induced damage and repair in nuclear and mitochondrial DNA in rat hippocampus and cortex. Two highly sensitive quantitative polymerase chain reaction (QPCR) assays were used to measure DNA damage. One assay measures the integrity of the entire mitochondrial genome and the other the integrity of nuclear DNA. The latter is a novel assay, developed in our laboratory, which utilizes the high copy number of short interspersed DNA elements (SINEs) residing in introns and untranslated regions of mammalian genes. A unique feature of the SINE-mediated QPCR is its ability to amplify simultaneously long random segments of DNA. Consequently, the SINE assay offers sufficient sensitivity for detecting DNA damage at levels that are compatible with the cellular capacity for DNA repair, and are likely to be consistent with cellular survival and therefore adequate for studying the DNA damage response in the brain. In rats, we found that exposure to an atmosphere of 4% oxygen for 30 min resulted in induction of DNA damage in nuclear and to a greater extent, in mitochondrial DNA. Following a 3-hr recovery period in ambient air, dissimilar repair kinetics for nuclear and mitochondrial DNA were measured.

Animals↗

Simple radioactive assay for the estimation of DNA breaks.

The intactness of DNA is an important part of the normal cellular structure. Any change to the DNA in the form of breaks leads to a change in the integrity, which in turn leads to abnormality in the cellular activity. Many discrepancies have been reported among the various methods of detecting DNA damage. Here, a simple, sensitive and reproducible method has been developed for the detection of DNA breaks by radioactive labelling of 5' broken ends. The method was evaluated by studying chemically induced DNA damage by using both organochloride (2,4-dichlorophenoxyacetic acid and lindane) and organophosphorus (sevin and phosphamidon) compounds at different concentrations. Phosphamidon, one of the organophosphorus compounds studied, showed complete degradation of the DNA after treatment. Radioactive analysis of phosphamidon showed higher counts at the lowest concentration (20 microg) of the chemical when compared with the control (2752 scintillation counts per minute, scm). Studies on the chemically induced DNA breaks by radiolabelling revealed that the cumulative effect of the organophosphorus and organochloride compounds showed maximum counts in all the samples (the highest being 2904 scm) when compared with the organophosphorus and organochloride compounds studied separately (the highest being 1881 and 2260 scm, respectively). Radiolabelling studies on the blood samples of 23 pesticide workers by the newly developed assay showed a significant positive correlation (0.893) between the number of years of exposure and the scintillation counts. A maximum of 11,702 scm (for 18 years of exposure) and a minimum of 1682 scm (for 4 years of exposure) were recorded compared with 1253 scm for the negative control. This method can be used effectively for estimation of the DNA breaks, irrespective of its nature.

Agricultural Workers' Diseases↗

Development of ELISAs measuring the extent of TAFI activation.

OBJECTIVE: To date, quantitation of TAFI antigen levels has been mainly focused on "total" antigen levels and has been shown to yield ambiguous results because of the existence of different isoforms and various degrees of activation. Our objective was to develop assays that allow measuring the extent of TAFI activation. METHODS AND RESULTS: A variety of enzyme-linked immunosorbent assays (ELISAs) were evaluated for their preferential reactivity toward TAFI before and after activation, and toward the recombinantly expressed activation peptide. Three ELISAs with distinct reactivities were selected: recognizing either exclusively nonactivated TAFI, the released activation peptide, or exclusively TAFIa (activated TAFI). Evaluation of TAFI activation during clot lysis revealed that decreases of TAFI levels are associated with increases of the released activation peptide and TAFIa levels. In addition, antigenic measurement of TAFIa parallels activity measured by chromogenic assay. Analyzing plasma samples revealed that subjects with hyperlipidemia had significantly higher plasma levels of both the activation peptide (109.2 versus 95.5; P<0.001) and TAFIa (112.1 versus 103.3; P=0.03), and not of TAFI antigen (92.5 versus 87.9; P=0.07) (results in % of plasma pooled from normolipidemic subjects). CONCLUSIONS: ELISAs that allow to measure the extent of TAFI activation were developed. These ELISAs constitute more sensitive markers in studies on the relationship between TAFI and cardiovascular diseases.

Biomarkers↗

From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part I.

Atherosclerotic cardiovascular disease results in >19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs. The recognition of the role of the vulnerable plaque has opened new avenues of opportunity in the field of cardiovascular medicine. This consensus document concludes the following. (1) Rupture-prone plaques are not the only vulnerable plaques. All types of atherosclerotic plaques with high likelihood of thrombotic complications and rapid progression should be considered as vulnerable plaques. We propose a classification for clinical as well as pathological evaluation of vulnerable plaques. (2) Vulnerable plaques are not the only culprit factors for the development of acute coronary syndromes, myocardial infarction, and sudden cardiac death. Vulnerable blood (prone to thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) play an important role in the outcome. Therefore, the term "vulnerable patient" may be more appropriate and is proposed now for the identification of subjects with high likelihood of developing cardiac events in the near future. (3) A quantitative method for cumulative risk assessment of vulnerable patients needs to be developed that may include variables based on plaque, blood, and myocardial vulnerability. In Part I of this consensus document, we cover the new definition of vulnerable plaque and its relationship with vulnerable patients. Part II of this consensus document focuses on vulnerable blood and vulnerable myocardium and provide an outline of overall risk assessment of vulnerable patients. Parts I and II are meant to provide a general consensus and overviews the new field of vulnerable patient. Recently developed assays (eg, C-reactive protein), imaging techniques (eg, CT and MRI), noninvasive electrophysiological tests (for vulnerable myocardium), and emerging catheters (to localize and characterize vulnerable plaque) in combination with future genomic and proteomic techniques will guide us in the search for vulnerable patients. It will also lead to the development and deployment of new therapies and ultimately to reduce the incidence of acute coronary syndromes and sudden cardiac death. We encourage healthcare policy makers to promote translational research for screening and treatment of vulnerable patients.

Acute Disease↗

From vulnerable plaque to vulnerable patient: a call for new definitions and risk assessment strategies: Part II.

Atherosclerotic cardiovascular disease results in >19 million deaths annually, and coronary heart disease accounts for the majority of this toll. Despite major advances in treatment of coronary heart disease patients, a large number of victims of the disease who are apparently healthy die suddenly without prior symptoms. Available screening and diagnostic methods are insufficient to identify the victims before the event occurs. The recognition of the role of the vulnerable plaque has opened new avenues of opportunity in the field of cardiovascular medicine. This consensus document concludes the following. (1) Rupture-prone plaques are not the only vulnerable plaques. All types of atherosclerotic plaques with high likelihood of thrombotic complications and rapid progression should be considered as vulnerable plaques. We propose a classification for clinical as well as pathological evaluation of vulnerable plaques. (2) Vulnerable plaques are not the only culprit factors for the development of acute coronary syndromes, myocardial infarction, and sudden cardiac death. Vulnerable blood (prone to thrombosis) and vulnerable myocardium (prone to fatal arrhythmia) play an important role in the outcome. Therefore, the term "vulnerable patient" may be more appropriate and is proposed now for the identification of subjects with high likelihood of developing cardiac events in the near future. (3) A quantitative method for cumulative risk assessment of vulnerable patients needs to be developed that may include variables based on plaque, blood, and myocardial vulnerability. In Part I of this consensus document, we cover the new definition of vulnerable plaque and its relationship with vulnerable patients. Part II of this consensus document will focus on vulnerable blood and vulnerable myocardium and provide an outline of overall risk assessment of vulnerable patients. Parts I and II are meant to provide a general consensus and overviews the new field of vulnerable patient. Recently developed assays (eg, C-reactive protein), imaging techniques (eg, CT and MRI), noninvasive electrophysiological tests (for vulnerable myocardium), and emerging catheters (to localize and characterize vulnerable plaque) in combination with future genomic and proteomic techniques will guide us in the search for vulnerable patients. It will also lead to the development and deployment of new therapies and ultimately to reduce the incidence of acute coronary syndromes and sudden cardiac death. We encourage healthcare policy makers to promote translational research for screening and treatment of vulnerable patients.

Animals↗

Comparison of the cyclooxygenase-1 inhibitory properties of nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors, using sensitive microsomal and platelet assays.

Two forms of cyclooxygenase (COX) activity are involved in the synthesis of prostaglandins, prostacyclins, and thromboxanes in mammalian cells. There is now convincing evidence, obtained with a number of structurally distinct inhibitors, that selective COX-2 inhibitors possess anti-inflammatory effects with an improved gastrointestinal tolerability compared with conventional nonsteroidal anti-inflammatory drugs (NSAIDs) affecting both COX-1 and COX-2. As more selective COX-2 inhibitors are being developed, assays with a high degree of sensitivity to inhibition are needed to compare the relative effects of compounds on COX-1 activity. In the present report, we describe a sensitive assay for the inhibition of human COX-1 based on the production of prostaglandin E2 by microsomes from U937 cells incubated with a subsaturating concentration of arachidonic acid. More than 45 NSAIDs and selective COX-2 inhibitors were tested in this assay. IC50 values ranged from 1 nM for flunixin and flurbiprofen to about 200-500 microM for salicylate and acetaminophen. Potent and nonselective NSAIDs such as sulindac sulfide, diclofenac, and indomethacin showed IC50 values of < 20 nM. Among the compounds that have been reported to show selectivity for COX-2, the rank order of potency against COX-1 was DuP 697 > SC-58451 > celecoxib > nimesulide-meloxicam-piroxicam-NS-398-RS-57067 > SC-57666 > SC-58125 > flosulide > etodolac > L-745,337 > DFU-T-614, with IC50 values ranging from 7 nM to 17 microM. A good correlation was obtained between the IC50 values for the inhibition of microsomal COX-1 and both the inhibition of TXB2 production by Ca2+ ionophore challenged platelets and the inhibition of prostaglandin E2 production by CHO cells stably expressing human COX-1. However, the microsomal assay was more sensitive to inhibition than cell-based assays and allowed the detection of inhibitory effects on COX-1 for all NSAIDs and selective COX-2 inhibitors examined with discrimination of their potency under conditions of limited availability of arachidonic acid.

Animals↗

Detection and quantification of antibodies to the extracellular domain of P0 during experimental allergic neuritis.

Quantification of the peripheral nerve myelin glycoprotein P0 and antibodies to P0 is difficult due to insolubility of P0 in physiological solutions. We have overcome this problem by using the water-soluble recombinant form of the extracellular domain of P0 (P0-ED) and describe newly developed assays which allow detection and quantitation of P0 and antibodies to P0, in serum and cerebrospinal fluid (CSF). These sensitive and specific assays based on the ELISA technique were used to study humoral immune responses to P0 during experimental autoimmune ("allergic") neuritis (EAN). In order to establish these tests, monoclonal antibodies to different epitopes of rodent and human P0-ED were produced. A two-antibody sandwich-ELISA allowing quantitation of P0 (lower detection limit of 0.5 ng/ml or 30 fmol/ml) and an antibody-capture ELISA (lower detection limit 1 ng specific antibody/ml) to detect antibodies to P0 in serum and CSF were developed. EAN was induced in rats by active immunization with bovine myelin or the neuritogenic protein P2 or by adoptive transfer using P2 specific CD4 positive T cells. Serum and CSF were assayed for the presence of P0-ED and antibodies to P0-ED or P2. Antibodies to P0-ED were detected during active myelin-induced EAN, but not during P2-induced or adoptive transfer EAN. The anti-P0-ED antibodies in the CSF showed a correlation with disease activity. In contrast, in the same model antibodies to P2 persisted long after the disease ceased. No soluble P0-like fragments could be found in serum or CSF during any of the three types of EAN. We conclude that P0 may be a B-cell epitope in EAN. These findings warrant a screen for antibodies to P0-ED in human immune neuropathies.

Animals↗

Immunoassay of total prostate-specific antigen using europium(III) nanoparticle labels and streptavidin-biotin technology.

Nanoparticle labels conjugated with biomolecules are used in a variety of different assay applications. We investigated the possibility of using europium(III)-labeled 68-nm nanoparticles coated with monoclonal antibodies or streptavidin (SA) to detect prostate-specific antigen (PSA) in serum. The selection of a suitable antibody pair and interference caused by the combination of nanoparticle label and structurally complex analyte were of special interest. A set of antibodies recognizing different epitope areas of PSA was mapped to find the optimal antibody pair for the immunometric nanoparticle-based assay. Different assay configurations were tested to obtain a good correlation with a conventional method based on biotinylated detection antibodies and europium(III) chelate-labeled streptavidin. Monoclonal capture antibody 5E4 was covalently coated on a microtitration well surface; biotinylated 5H6 monoclonal antibody (Mab) was used for detection, and europium(III)-labeled streptavidin-coated nanoparticles were utilized for signal generation. Total PSA concentrations were determined from a panel of male serum samples to test the developed assay. The correlation of the nanoparticle-based and reference assays was good; y=0.9844x-0.1252, R2=0.98, n=27; and the lowest limit of detection of the assay (LLD=0.83 ng/l) was 35-fold lower than for the reference method. The assay application presented here, where a structurally complex analyte is detected, combines the exceptionally high affinity of streptavidin-biotin technology and the high specific activity of long lifetime fluorescence nanoparticle labels. The general characteristics of this combination should permit the development of various immunoassay applications featuring high sensitivity, rapidity, and low consumption of reagents.

Antibodies, Monoclonal↗

Accuracy of enzyme-linked immunosorbent assay using crude and purified antigens for serodiagnosis of melioidosis.

Five enzyme-linked immunosorbent assays developed to detect antibodies to different Burkholderia pseudomallei antigen preparations were evaluated as diagnostic tests for melioidosis in northeast Thailand. The highest diagnostic indices were observed for an affinity-purified antigen (sensitivity, 82%; specificity, 72%) and crude B. pseudomallei antigen (sensitivity, 81%; specificity, 70%), an improvement over the indirect hemagglutination assay (sensitivity, 73%; specificity, 64%).

Antibodies, Bacterial↗

Quantitative assays for hepatitis C virus in serum as predictors of the long-term response to interferon.

BACKGROUND/AIMS: Interferon therapy has a beneficial effect in patients with chronic hepatitis C who have a low viral load. The aim of this study was to compare the core protein level with HCV RNA levels and to analyze whether virus quantitation predicts the efficacy of interferon therapy. METHODS: HCV core protein level assessed by the recently developed assay was compared with HCV RNA levels measured by three different methods (Amplicor-HCV monitor, competitive RT(CRT)-PCR, and bDNA probe assay) in 352 patients with chronic hepatitis C in relation to viral serotype. RESULTS: From 91% (320/352) to 93% (299/322) of patients with viremia were detected by Amplicor-monitor and CRT-PCR, in contrast to 60% (187/312) and 74% (191/258) by bDNA and HCV core protein assay, respectively. The HCV core protein level was positively correlated with HCV RNA levels measured by the three assays (r = 0.680 to 0.731). Serum HCV RNA and core protein levels were significantly lower in patients with serotype 2 than in those with serotype 1. Viral eradication after interferon therapy was observed in 60-70% of the patients with < 1 x 10(4) copies/ml of HCV RNA by Amplicor-monitor assay, < 2 x 10(5) copies/ml by CRT-PCR, < 0.5 Meq/ml by bDNA assay, and < 20 pg/ml of core protein by HCV core protein assay. Viral eradication was uncommon (< 11%) among the patients with higher viral loads. Bivariate analysis revealed that the outcome of interferon therapy was more closely associated with both HCV core protein and RNA levels than the HCV serotype. CONCLUSIONS: Quantitation of HCV core protein and HCV RNA in useful for prediction of the interferon response.

Adolescent↗

[Epidemiologic characteristics of nutrition of school children at high risk for developing ischemic heart disease and atherosclerosis].

The results of the investigation of a representative sample of Moscow schoolchildren, aged 11-14 years, are described. The investigation included a triple arterial pressure measuring, anthropometry, numerical score of sexual development, assay of cholesterol, triglycerides and high-density lipoprotein cholesterol levels in the blood serum. The character of nutrition of 250 (20% of the sample) schoolchildren was studied by the method of dietary inquiry (daily ration) with the use of food patterns. The data of the multivariative analysis have shown that sexual development, a low educational level of the parents (among them subjects engaged in the manual labor being predominant) influence the development of the risk factors of ischemic heart disease and atherosclerosis. Composition of food is of great importance, especially for boys with dislipoproteinemias (low consumption of starch per 1 kg bw, high consumption of total protein, low consumption of fat in cal %), and for those with excessive body mass and low physical activity (low consumption of total fat, polyunsaturated fat per 1 kg bw, as well as high consumption of total protein in cal %).

Adolescent↗

Validation of an HPLC method for the determination of CP-93,393 in CP-93,393-1 tablets.

A reversed-phase high performance liquid chromatographic (HPLC) assay developed for the CP-93,393-1 drug substance was adapted for use with CP-93,393-1 tablets. Using a novel experimental matrix, validation was performed to obtain linearity, reproducibility and recovery and to meet current regulatory requirements. Deviations in the sample preparation procedure were performed to demonstrate the ruggedness of the assay.

Anti-Anxiety Agents↗

Development of a novel trans-lentiviral vector that affords predictable safety.

Lentiviral vectors derived from human immunodeficiency virus type 1 (HIV-1) hold great promise for gene therapy. However, the possibility of generating replication-competent retrovirus (RCR) through genetic recombination raises concerns for safety. Here we describe a novel HIV-based packaging system (trans-lentiviral) that splits gag/gag-pol into two parts: One that expresses gag/gag-pro and another that expresses reverse transcriptase and integrase as fusion partners of viral protein R (Vpr). Using a sensitive assay developed to specifically detect recombinant lentiviral DNA mobilization, we demonstrated that the trans-lentiviral vector prevents the generation of recombinants that contain a functional gag-pol structure, while the lentiviral vector generates env-minus recombinant lentivirus that mobilizes recombinant genomes to other cells when pseudotyped with an exogenous envelope. Since an intact gag-pol structure is absolutely required for retroviral DNA mobilization and RCR, the trans-lentiviral vector design significantly reduces this risk. Moreover, it makes it possible to assess the risk of RCR and DNA mobilization using an in vitro assay that monitors trans-lentiviral vector stocks for the regeneration of the gag-pol structure. Therefore, the trans-lentiviral vector design will ensure the greatest predictable level of safety for the clinical application of retroviral vectors, including HIV-based vectors.

Antigens, CD34↗

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk.

We have developed a quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment from a wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCR products generated using a digoxigenin-labelled primer were heat-denatured before being quantified by an enzyme-linked immunosorbent assay (ELISA). A biotinylated probe immobilized onto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbency differences when assaying milk samples containing bacteria in the range 10(3)-10(7) cfu ml-1. The detection threshold for the PCR-ELISA assay developed in this work is 103 cfu ml-1.

Animals↗

Spectrofluorimetric quantification of malondialdehyde for evaluation of cyclooxygenase-1/thromboxane synthase inhibition.

The in vitro assay developed by Hartmann and Ledergerber (1995) utilizing the spectrofluorimetric quantification of malondialdehyde after reaction with thiobarbituric acid was modified and used for further investigations. The human whole blood was replaced by a platelet suspension of pig blood, and calcium ionophore A23187 was used instead of collagen for inducing the arachidonic acid cascade. The modified assay represents a simple, time and cost saving method for the evaluation of cyclooxygenase-1/thromboxane synthase inhibition. The reproducibility and comparability of results is given. Additional experiments allow classification of selective phospholipase A2, cyclooxygenase-1, and thromboxane synthase inhibitors. Further studies of malondialdehyde formation show that the cyclooxygenase and/or the thromboxane synthase are competitively inhibited by reaction products of the cyclooxygenase pathway by a negative feedback mechanism.

Animals↗

An improved measurement of progesterone in saliva and clinical applications.

Measurement of progesterone in saliva offers several advantages when compared to assays of serum progesterone, especially when ovarian activity is being assessed. Most published methods for the determination of progesterone in saliva are based on assays developed in research centers, which employ "in-house" reagents that are critically dependent on supplies of highly selected antisera. In this report, the adaptation of a readily available commercial progesterone "kit", the Pantex Immunodirect Progesterone (125I) is described for the measurement of salivary progesterone. A single extraction step was added, however, to improve assay performance and to ensure that total salivary progesterone was measured.

Female↗

Comparison of a salivary/sputum pepsin assay with 24-hour esophageal pH monitoring for detection of gastric reflux into the proximal esophagus, oropharynx, and lung.

The purpose of this study was to determine whether measurement of salivary/sputum pepsin could be used as a surrogate marker for detecting gastroesophageal reflux using 24-hr esophageal pH monitoring as the gold standard. Patients with gastroesophageal reflux symptoms underwent simultaneous 24-hr esophageal pH monitoring and collection of saliva and sputum samples for pepsin measurement using a recently developed assay. In all, 16 patients provided 19 positive (10.6%) and 161 negative pepsin assays. The mean pH values for the positive pepsin samples were lower then the negative samples at both the proximal [5.34 (95% CI, 4.94-5.75) vs 6.12 (95% CI, 6.03-6.20; P < 0.01)] and distal [4.97 (95% CI, 4.61-5.33) vs 6.03 (95% CI, 5.92-6.15; P < 0.01)] pH probes. Proximal esophageal reflux was not detected in patients who had a negative pepsin assay (N = 12); in contrast, proximal esophageal reflux was documented in three of four patients with a positive assay. In conclusion, detection of pepsin in the saliva and/or sputum may provide a noninvasive method to test for the proximal reflux of gastric contents.

Esophagoscopy↗