Enzyme Immunosorbent Assay Research Articles

Increased expression of NF-AT3 and NF-AT4 in the atria correlates with procollagen I carboxyl terminal peptide and TGF-β1 levels in serum of patients with atrial fibrillation.

BackgroundAtrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice. Unfortunately, the precise mechanisms and sensitive serum biomarkers of atrial remodeling in AF remain unclear. The aim of this study was to determine whether the expression of the transcription factors NF-AT3 and NF-AT4 correlate with atrial structural remodeling of atrial fibrillation and serum markers for collagen I and III synthesis.MethodsRight and left atrial specimens were obtained from 90 patients undergoing valve replacement surgery. The patients were divided into sinus rhythm (n = 30), paroxysmal atrial fibrillation (n = 30), and persistent atrial fibrillation (n = 30) groups. NF-AT3, NF-AT4, and collagen I and III mRNA and protein expression in atria were measured. We also tested the levels of the carboxyl-terminal peptide from pro-collagen I, the N-terminal type I procollagen propeptides, the N-terminal type III procollagen propeptides, and TGF-β1 in serum using an enzyme immunosorbent assay.ResultsNF-AT3 and NF-AT4 mRNA and protein expression were increased in the AF groups, especially in the left atrium. NF-AT3 and NF-AT4 expression in the right atrium was increased in the persistent atrial fibrillation group compared the sinus rhythm group with similar valvular disease. In patients with AF, the expression levels of nuclear NF-AT3 and NF-AT4 correlated with those of collagens I and III in the atria and with PICP and TGF-β1 in blood.ConclusionsThese data support the hypothesis that nuclear NF-AT3 and NF-AT4 participates in atrial structural remodeling, and that PICP and TGF-β1 levels may be sensitive serum biomarkers to estimate atrial structural remodeling with atrial fibrillation.

Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the highest activity of systemic lupus erythematosus. Our results show that native circulating IgG complexes from active systemic lupus erythematosus patients expose fucosyl residues and their glycan core is accessible to soluble lectins. Two putative mechanisms may contribute to the increased exposure of these glycans: (1) the canonical N-glycosylation site of the IgG-CH2 domain; (2) an IgG binding non-IgG molecule, like complement or C-reactive protein. In both cases the complexed IgG may be alternatively targeted to lectin receptors of effector cells, e.g. dendritic cells.

Host response biomarker in sepsis: suPAR detection.

Recent studies of our group have shown that suPAR may complement APACHE II score for risk assessment in sepsis. suPAR may be measured in serum of patients by an enzyme immunosorbent assay developed by Virogates (suPARnostic™). Production of suPAR from circulating neutrophils and monocytes may be assessed after isolation of neutrophils and monocytes and ex vivo culture. This is followed by measurement of suPAR in culture supernatants.

Adropin: a key component and potential gatekeeper of metabolic disturbances in policystic ovarian syndrome

Purpose: The aim of the current study was to evaluate potential relationships between serum adropin levels and metabolic parameters in polycystic ovary syndrome (PCOS). Materials and Methods: Twenty women with PCOS and 20 healthy, age and body mass index (BMI) matched controls were included in the study. All subjects underwent venous blood drawing on the early follicular phase after an overnight fasting. Serum adropin levels were measured with enzyme immunosorbent assay (EIA). The relationships between serum adropin levels and metabolic parameters were also assessed. Results: Serum adropin levels were found to be significantly lower in women with PCOS when compared to control group (p Conclusion: The findings of current study suggest that women with PCOS have low serum adropin levels that may contribute to the underlying pathogenesis of metabolic disturbances in PCOS.

AB0556 Monocyte Chemoattractant Protein-1 in Systemic Lupus Erythematosus

BackgroundIn recent years, potential biological markers of systemic lupus erythematosus (SLE) and LN activity have been intensely sought for. Among them, it seems that monocyte chemoattractant protein-1 (MCP-1) has a...

Interleukin-1β genotype and circulating levels in cancer patients: Metastatic status and pain perception

ObjectivesProinflammatory cytokines released during inflammation can cause hyperexcitability in pain transmission neurons, leading to hyperalgesia and allodynia. Polymorphisms in interleukin 1 (IL-1) family of genes (IL1A, IL1B) and in IL-1 receptor antagonist (IL-1Ra, coded by IL1RN) may therefore induce alterations in cytokine levels/effects and pain related response. Our purpose was to investigate the influence of polymorphisms in IL1A/B/RN on cytokine serum levels and its correlation with pain intensity, performance status, adverse effects, metastases and breakthrough pain in Caucasian cancer patients. Design and methodsSerum IL-1α/β levels of 74 cancer patients were measured by competitive enzyme immunosorbent assay. All patients were also genotyped for the polymorphisms in IL1A (rs17561), IL1B (rs1143634) and IL1RN (rs419598) with Real-Time PCR. Results were then correlated to the appearance of bone or CNS metastases and several pain-related parameters. ResultsIL-1β rs1143634 homozygous for T allele were associated with lower levels of IL1-β (p=0.032, Mann–Whitney test) and presented a trend for lower levels of pain (p=0.06, Fisher's Exact Test). Also, IL1-β levels were related with cancer onset status, since a four-fold increase probability of metastatic disease was observed in high IL-1β individuals (OR=4.074, p=0.010, Pearson χ2 test). Among the female patients presenting metastatic disease and carriers of the TT genotype we observed a trend to lower levels of IL1-β (p=0.053, Pearson χ2 test). ConclusionsOur results indicate that genetic variation at IL1-β gene may influence serum levels of IL1-β, with proportional consequences in cancer-related pain.

Influence of Probiotic Consortium on TH1 and TH2 Immune Response.

IntroductionThe main role of probiotics is to maintain homeostasis in the intestines and improve bowel protective function. The aim of the present study is to investigate immuno-modulatory effects of a probiotic consortium.MethodsObservations were carried out in vitro. The presence of IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, IgA, IgM, and IgE was studied using a solid-phase enzyme immunosorbent assay on the VECTOR-BEST sets (Russia).ResultsImmunomodulatory properties of the probiotic consortium were studied, which consisted of the following strains: Streptococcus thermophilus, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus acidophilus, Bifidobacterium longum, and Bifidobacterium bifidum. Elevated concentrations of INFγ in control samples decreased 3.9 times (p < 0.05) after a saturation of blood with the probiotic consortium. Significant reduction of cytokine levels after the probiotic effects of the consortium was observed in IL-10 by 2.1 times (p < 0.05) and IgA by 1.87 times (p < 0.0005). There was a significant increase in the levels of IL-4, IgE, IL-6, and IL-8 by 1.3 (p < 0.005), 1.1 (p < 0.5), 18.0 (p < 0.005), and 6 (p < 0.05) times, respectively, in comparison with the control samples. IL-4 and INFγ have different effects on the synthesis of IgE. Soluble low affinity receptors FcɛRII (CD23) in association with IL-4 facilitate a differentiation of the B-lymphocytes in IgE-synthesizing cells, while γ-INF inhibits this process. It is known that the intracellular expression of γ-INF and IL-4 is the most reliable marker for Th1 and Th2 immune responses, respectively. The conducted studies determined that the ratio of INF-γ/IL-4 was 0.9 (control 4.8, P < 0.005) after the saturation of the blood cells with probiotic consortium. NF-γ/IL4 ratio decreased by 5.3 times compared with a control value, which indicates a reduction in the functional activity of Th1 type lymphocytes in comparison with the function of Th2 cells.ConclusionThe application of the probiotic consortium results in the maintenance of homeostasis by the stimulation of immune function through the activation of humoral immunity. Moreover, the probiotic application changes the orientation of the immunological memory causing the cancellation of the recruitment of Th1 cells in the response.

Expression of SDF-1 and CXCR4 transcript variants and CXCR7 in epithelial ovarian cancer.

Chemokine stromal cell-derived factor-1 (SDF-1) and its receptors, CXCR4 and CXCR7, have been implicated in epithelial ovarian cancer progression and metastasis. However, limited data are available on the expression levels of SDF-1 and CXCR4 variants and CXCR7 in human epithelial ovarian cancer. The present study aimed to characterize the expression pattern and levels of SDF-1, CXCR4 and CXCR7 in normal human ovaries and epithelial ovarian cancer. The expression of SDF-1 and CXCR4 transcript variants and CXCR7 was determined by quantitative polymerase chain reaction (qPCR). Plasma SDF-1α levels were determined by commercially available EIA kits and cancer antigen 125 (CA 125) levels were quantified by automated microparticle enzyme immunosorbent assay. High expression levels of SDF-1 transcript variant 1 were identified in ovarian cancer and control ovaries. By contrast, in both groups the expression levels of SDF-1 transcript variants 3 and 4 were extremely low. Furthermore, SDF-1 variant 1 levels were notably higher in epithelial ovarian cancer than in control ovaries, while data for the remaining transcripts were similar in both groups. CXCR4 transcript variant 2 and CXCR7 expression levels in normal and neoplastic ovaries were similar. In both groups, CXCR4 transcript variant 2 was not detected. Plasma SDF-1α levels were notably higher in females with epithelial ovarian cancer than in the control ovaries. Elevated levels of blood SDF-1α were found prior to surgery, 6 days after surgery and following completion of the first chemotherapy course. These increases were independent of the type of epithelial ovarian cancer. Our results suggest that the expression of SDF-1 and the genes controlling alternative splicing are elevated in epithelial ovarian cancer, leading to an increased formation of SDF-1 variant 1. Elevated plasma SDF-1α levels in epithelial ovarian cancer patients are not associated with the presence of tumors and/or metastases, however reflect a general response to the disease.

Exhaled IL-8 in Systemic Lupus Erythematosus with and without Pulmonary Fibrosis

The purpose of this study is to evaluate the relationship between the concentration of interleukin-8 (IL-8) in exhaled breath condensate (EBC) and bronchoalveolar lavage fluid (BALF) with the disease activity score and pulmonary function of systemic lupus erythematosus (SLE) patients with and without pulmonary fibrosis. Thirty-four SLE patients and 31 healthy controls were enrolled and evaluated using high-resolution computed tomography (HRCT), pulmonary function tests, systemic lupus activity measure (SLAM), assessing BALF and EBC. IL-8 levels in BALF and EBC samples were measured with an enzyme-immunosorbent assay kit. The mean (±SEM) IL-8 concentrations in BALF and EBC were higher in SLE patients compared to healthy controls (34.84 ± 95.0 vs. 7.65 ± 21.22 pg/ml, p < 0.001; 3.82 ± 0.52 pg/m vs. 1.7 ± 1.7 pg/ml, p < 0.001, respectively). SLE patients had increased percentage of neutrophils in BALF when compared with control group (1.00 ± 5.99 vs. 0.00 ± 0.56 %, p = 0.0003). Pulmonary fibrosis in HRCT was found in 50 % of SLE patients. The disease activity scored by SLAM was significantly higher and total lung capacity was significantly lower in SLE patients with pulmonary fibrosis (8.00 ± 3.17 vs. 6.00 ± 2.31, p = 0.01; 88.00 ± 28.29 vs. 112.00 ± 21.08 % predicted, p = 0.01, respectively). In SLE patients with pulmonary fibrosis, correlations were found between SLAM and IL-8 concentration in BALF, forced expiratory volume in 1 s and forced vital capacity (r = 0.65, p = 0.006; r = −0.53, p = 0.035; r = −0.67, p = 0.006, respectively). Our results indicate that IL-8 plays an important role in the pathogenesis of SLE. An increased concentration of IL-8 according to BALF could be considered as a useful biomarker of SLE activity and pulmonary fibrosis in SLE.

Molecular characteristics of rotavirus isolated from a diarrhea outbreak in october 2008 in bintuni bay, papua, indonesia.

BACKGROUNDViral diarrhea continues to be a health problem in Indonesia that often causes outbreaks; in particular, acute viral diarrhea in young children. Rotavirus is the leading cause of severe diarrhea in children under two years of age. This study aimed to determine the genotypes of rotavirus in Bintuni Bay, Papua.METHODSStool specimens from 15 patients were collected and analyzed for rotavirus using an enzyme immunosorbent assay (EIA) and reverse transcriptase-polymerase chain reaction (RT-PCR). Subsequently, we sequenced the genetic material of rotavirus positive samples by RT-PCR and analyzed the results using Mega-4 software.RESULTSTwo rotavirus serotypes were identified from the diarrhea outbreak in Bintuni, Papua in October 2008: serotype G1 with G1P[6] (50%) and G1P[8] (16.7%) strains, and serotype G2 with G2P[4] (23.3%) strain. Phylogenetic tree analyses of VP7 protein showed that rotavirus-infected diarrhea in Bintuni Bay, Papua at that time was dominated by the G1 serotype (83%).CONCLUSIONThe laboratory results showed that G1 serotype rotavirus was a cause of the outbreak of diarrhea in October 2008 in Bintuni, Papua.

Evaluation of a chromogenic culture medium for the detection of Clostridium difficile.

PurposeClostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI.Materials and MethodsIn efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection.ResultsChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h.ConclusionSimultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI.

CALCITONIN GENE-RELATED PEPTIDE IN SERUM AFTER SPONTANEOUS SUBARACHNOID HEMORRHAGE

Endogenous Calcitonin Gene-Related Peptide (CGRP) stored in perivascular nerve fibers around the cerebral arteries mediates vasodilation and regulates cerebral blood flow under physiological conditions. Increased levels of CGRP have been continually reported in patients with migraine, but the role of CGRP in the pathophysiology of Subarachnoid Hemorrhage (SAH) has not yet been sufficiently evaluated. Over 10 days, serum was prospectively collected from 33 consecutive patients (17 women, 16 men; mean age 52.8 years) with spontaneous non-traumatic SAH. All patients were graded III or IV according to the Fisher Score. CGRP levels were determined by means of an Enzyme-Immunosorbent Assay (EIA). CGRP concentrations were correlated to (1) the anatomical localization of the aneurysm (anterior circulation or posterior circulation), (2) the treatment modality (clipped group, coiled group, or untreated group) and (3) the rate of ischemia determined by computed tomography (ischemia group, non-ischemia group, or iatrogenic ischemia group). (1) Anatomical localization: CGRP levels were significantly higher in patients with a ruptured aneurysm of the anterior circulation (p<0.001). (2) Treatment modality: CGRP levels differed significantly between the groups (coil group, clip group, or untreated group; p<0.001). The highest levels of CGRP were detected in the coil group and the lowest levels in the untreated group. About (3) Ischemia: CGRP levels differed significantly between the three groups (ischemia group, non-ischemia group, or iatrogenic ischemia group; p = 0.038). The highest levels of CGRP were found in the ischemia group. The maximum levels of CGRP in serum were found for patients with a ruptured aneurysm of the anterior circulation and subsequent ischemia treated by endovascular coiling. These findings support the theory that CGRP is produced and excessively released to counteract cerebral vasospasm and subsequent ischemia. Presumably, the serum levels of CGRP are affected by the anatomical localization of the aneurysm and can be stimulated by any endoluminal manipulation of the parent artery.

A comparative clinical study of efficacy of microimmuno assay with WIDAL-test in enteric fever in children

The diagnosis of typhoid fever in young children is also a dilemma because of its manifestations and typical presentation may not be seen in all cases. Antibodies to Salmonella typhi antigen are developed in the human body, which can be detected as a diagnostic test for the enteric fever. Objective: This study was undertaken to compare the efficacy of WIDAL-test with micro-immunoassay (dot enzyme immunosorbent assay). Method: 40 cases of clinically suspected enteric fever cases were included in this study. Result: In the present study, nearly 92% were positive for micro immunoassay (dot-enzyme immunosorbent assay) by Enterocheck-WB kit, 80% were positive for WIDAL and only 15% were culture positive. Immunoassay positive, but WIDAL negative cases were 20%, whereas WIDAL positive and immunoassay negative cases were only 7.5%. The positive predictivity of micro-immunoassay in diagnosing enteric fever is better than WIDAL both in 1st and 2nd week of illness. Micro-immunoassay done in the study was rapid in diagnosing the case. Conclusion: It is concluded from the present study that the micro-immunoassay (Enterocheck-WB) is better than WIDAL-test in the diagnosis of enteric fever in children.

Laying Hen’s Primary Immune Response to Staphylococcal Protein A (SpA)

Our aim was to provide information about the production of Egg White Immunoglobulin (EWIg) with specificity to Staphylococcal protein-A, a surface antigen of Staphylococcus aureus and to study the inhibition of this bacterium growth in pre- and post-immunized hens. A sandwich Enzyme-Immunosorbent Assay (ELISA) showed a large concentration of anti-SpA antibodies in the eggs from hens immunized with protein A. The titer of these antibodies was at least 5 to 6-folds of that of the eggs from pre-immunized hens 10 days post-immunization. Inhibition of the growth of S. aureus by anti-SpA antibodies purified by SpA-affinity chromatography (PURE-1A) was observed in laying hens vaccinated. Growth of the bacteria in blood agar plates occurred in antibody samples from preimmunized laying hens only. Inhibition of the agglutination of SpA-bearing Staphylococcus aureus cells by purified anti-SpA antibodies was observed in vitro. The authors are not aware of previous studies of the primary immune system response developed in eggs from laying hens, so this research could set a precedent in the field of egg white immunoglobulin technology. The use of hyper-immune eggs as alternative to the use of antibiotics could be advantageous for the large amount of antibodies produced, low cost, the reduction of antigenic variation and very low toxicity.

Seroprevalence of parvovirus B19 IgG and IgM antibodies among pregnant women in Oyo State, Nigeria

Human parvovirus B19 causes a wide range of complications in pregnant women including abortion, severe fetal anemia, non-immune hydrops fetalis, and even intrauterine fetal death. However, there is a dearth of information on the prevalence of the virus among pregnant women in southwestern Nigeria. Blood samples were collected from 231 pregnant women and screened for antibodies to human parvovirus B19 IgM and IgG using an enzyme immunosorbent assay kits. Of the 231 women, 31 were in their first trimester, 146 were in their second trimester, and 54 were in their third trimester. Forty-five (20%) were positive for parvovirus B19 IgG antibodies, 10 (4%) were positive for parvovirus B19 IgM antibodies, and 176 (76%) had no detectable parvovirus B19 antibodies. Twenty-eight (19%) of the 146 pregnant women in their second trimester were positive for parvovirus B19 IgG antibody while three (2%) of the 146 were positive for parvovirus B19 IgM antibody. It is evident that there is a high prevalence of human parvovirus B19 among pregnant women in south-western Nigeria. This suggests that there is an active transmission of the virus in the community; it is therefore necessary to conduct more studies on the virus in pregnant women in Nigeria to ascertain its effect on the fetus.

Glia and epilepsy: Experimental investigation of antiepileptic drugs in an astroglia/microglia co‐culture model of inflammation

The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia-mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co-culture model. Primary astrocytic cultures were prepared from brains of postnatal (P0-P2) Wistar rats and co-cultured with a physiologic amount of 5%, as well as 30% microglia in order to mimic inflammatory conditions. Co-cultures were treated with valproic acid (VPA), carbamazepine (CBZ), phenytoin (PHE), and gabapentin (GBT). Viability and proliferation were measured using the tetrazolium (MTT) assay. The microglial activation state was determined by immunocytochemical labeling. The astroglial connexin 43 (Cx43) expression was measured by Western blot analysis. The transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) cytokine levels were measured by the quantitative sandwich enzyme immunosorbent assay (ELISA). Astrocytes, co-cultured with 5% microglia (M5 co-cultures), showed a dose-dependent, significant reduction in glial viability after incubation with PHE and CBZ. Furthermore, VPA led to highly significant microglial activation at all doses examined. The antiinflammatory cytokine TGF-β1 release was induced by high doses of GBT and PHE. Astrocytes co-cultured with 30% microglia (M30 co-cultures) revealed a dose-dependent significant reduction in glial viability after incubation with PHE, accompanied by increased TGF-β1 and TNF-α levels. However, CBZ significantly reduced the amount of activated microglial cells and increased the total number of inactivated microglia. Finally, CBZ resulted in reduced viability at all doses examined. CNS inflammation is characterized by a disturbance of glial cell functions. Strong microglial activation, a typical hallmark of inflammation, was induced by VPA in M5 and continued in M30 co-cultures. With regard to the direct relation between CNS inflammation and seizures, VPA seems to be unsuitable for reducing inflammatory conditions. The reverse effect was achieved after CBZ. We noticed significant microglial inactivation, after incubation of the M30 co-cultures. In conclusion, we suggest that AEDs with antiinflammatory glial features are beneficial for seizures caused by persistent brain inflammation.

An automatic enzyme immunoassay based on a chemiluminescent lateral flow immunosensor

Microfluidic integrated enzyme immunosorbent assay (EIA) sensors are efficient systems for point-of-care testing (POCT). However, such systems are not only relatively expensive but also require a complicated manufacturing process. Therefore, additional fluidic control systems are required for the implementation of EIAs in a lateral flow immunosensor (LFI) strip sensor. In this study, we describe a novel LFI for EIA, the use of which does not require additional steps such as mechanical fluidic control, washing, or injecting. The key concept relies on a delayed-release effect of chemiluminescence substrates (luminol enhancer and hydrogen peroxide generator) by an asymmetric polysulfone membrane (ASPM). When the ASPM was placed between the nitrocellulose (NC) membrane and the substrate pad, substrates encapsulated in the substrate pad were released after 5.3±0.3min. Using this delayed-release effect, we designed and implemented the chemiluminescent LFI-based automatic EIA system, which sequentially performed the immunoreaction, pH change, substrate release, hydrogen peroxide generation, and chemiluminescent reaction with only 1 sample injection. In a model study, implementation of the sensor was validated by measuring the high sensitivity C-reactive protein (hs-CRP) level in human serum.

Absence of anti‐hepatitis B virus (HBV) core in HIV/HBV coinfection with advanced immunosuppression

Hepatitis B virus (HBV) coinfection is frequent among HIV-infected patients and is associated with poorer outcomes related to monoinfection with either virus [1]. These patients may present atypical HBV serological patterns, such as nonreactive anti-HBV core (HBc) with reactive HBV surface antigen (HBsAg) [2]. We investigated the prevalence of HBsAg with nonreactive anti-HBc in a cohort of HBV/HIV-coinfected patients from an AIDS Clinic in Sao Paulo, Brazil. The study population was selected from a cohort of 2412 HIV-positive patients, of whom 120 were HBsAg reactive. Cases were chronically HBV-infected patients with nonreactive anti-HBc, either transiently or persistently. Controls were obtained from the same population, but had persistently reactive anti-HBc. Cases comprised 11 men and one woman (39 - 61 years old), while controls comprised 24 anti-HBcand HBsAg-reactive patients matched by age and gender in a control:case ratio of 2:1. HIV results were obtained using two commercial enzyme immunosorbent assays (Organon Technika, Tournault, Belgium and Embrabio, Sao Paulo, Brazil) and confirmed using GS-HIV-1 Western Blot (Bio-Rad, Hercules, CA). HIV RNA was quantified using the Versant HIV-1 RNA 3.0 bDNA assay (Siemens Healthcare, Erlangen, Germany). CD4 T-lymphocyte counts were determined using the BD Multitest/Trucount (BD Biosciences, San Jose, CA). A microparticle enzyme immunoassay (AxSYM; Abbott Laboratories, North Chicago, IL) was used to detect HBV serological markers. HBV DNA was quantified using real-time polymerase chain reaction [3] and pre-core/core and surface gene sequences were obtained for mutation detection and subgenotyping in HBV DNA-positive cases [4]. For each patient, the mean CD4 count and viral load measurement for all measurements performed 12 months prior to each HBV serological test were calculated. Opportunistic diseases and complications of chronic HBV infection were also reported. The use of HBV-active drugs (lamivudine, tenofovir, entecavir or interferon) prior to the first serological test included in the study for cases and at any time for controls was evaluated. Variables were compared using the two-sided Wilcoxon rank-sum test (for continuous variables) or the two-sided Fisher’s exact test (for categorical variables) with STATA 10.1 (Stata Corporation, College Station, TX), and a significance level of 0.05. We identified 120 (5.0%) chronically HBV-infected patients, 12 (10.0%) of whom presented with nonreactive anti-HBc. A significantly higher proportion of cases were persistently positive for HBeAg, and cases had a lower mean CD4 count compared with controls (Table 1). Amplification of HBV DNA was achieved in three out of seven cases who were available for collection of a new sample, with a mean viral load of 5.67 log HBV DNA copies/mL. HBV subgenotype A1 was detected in all three cases. No mutations in pre-core/core and S gene sequences were identified.

Identification and characterization of microRNAs in vascular smooth muscle cells from patients with abdominal aortic aneurysms

The role of microRNAs (miRs) in the development of various cardiovascular diseases was recently highlighted in several studies. However, the biological role of miRs in the pathogenesis of abdominal aortic aneurysms (AAAs) is still not well defined. The present study aims to identify and characterize miR expression in the aortic explant cultures of AAA patients. A new microarray platform (miChip) using locked nucleic acid-modified capture probes with increased sensitivity and specificity was employed to detect the miR expression profile in human vascular smooth muscle cell cultures from AAA surgical samples. The array data were further validated by real-time quantitative reverse transcription polymerase chain reaction (qPCR) experiments with additional AAA and control aorta samples. Potential target genes of the differentially expressed miRs were predicted by bioinformatics analysis. Some of the results were confirmed by enzyme immunosorbent assay and Western blot analysis. Total RNA extracted from the explant of six AAAs and six normal aortas exhibited notably different miR profiles. A twofold difference of miR-516a-5p and miR-1260 was expressed in AAAs compared with normal aortic cultures (P < .05). The expression of miR-516a-5p was more than three times higher in AAAs, and miR-1260 expression was almost four times lower, as validated by qPCR. Additional qPCR performing on an extra 10 AAAs and 10 control aortas showed similar results. The expression of three predicted targets--secretory interleukin-3, vascular endothelial growth factor A, and collagen type 1, alpha 1 proteins--was significantly elevated in aneurysmal cultures compared with normal aortic cultures (P < .05). This miR microarray study displayed an altered expression of miR-516a-5p and miR-1260 in AAAs compared with control aortas. Functional annotations of the two miRs via bioinformatics approaches revealed that both are highly involved in some predefined mechanisms of AAA formation. Three of their target genes were also upregulated in AAAs. The results may be critical to elucidate the functional role of miRs in diseased aorta.

Clinical significance of cytokines levels in exhaled breath condensate (EBC) and serum of lung cancer patients.

e22202 Background: Cytokines are known to play an important role in carcinogenesis.The aim of this study is to measure pre-treatment concentrations of cytokines IL6, IL10, IL1β and IL8, in EBC and serum of lung cancer patients and to assess any possible association with clinicopathological variables and survival. Methods: From February 2010 to May 2011 we enrolled 73 patients with previously untreated lung cancer, 67 males and 6 females, age 67.7±9.5 years, 46 smokers and 27 ex-smokers, and 30 healthy individuals as controls. Sixty-one patients had NSCLC and 12 SCLC (5 stage I, 3 stage II, 19 stage III and 46 stage IV). EBC was collected using the Ecoscreen device (Viasys, Germany). Serum and EBC levels of cytokines were analyzed by commercially available enzyme-immunosorbent assay kits (ELISA). Results: All cytokines but IL8 were detectable in both materials. IL8 was not detected in EBC. All cytokines were significantly increased in patients with lung cancer in comparison to controls (IL6: serum P&lt;0.0001, EBC P=0.001; IL10: serum P= 0.006, EBC P=0.037; IL1β: serum P= 0.032, EBC P=0.005; IL8 serum P=0.001). Moreover, a statistically significant correlation was found between IL10 levels in EBC and serum (P=0.001, Rho=0.334). In EBC, higher levels of IL6 were found in patients with T3-T4 disease than in those with T1-T2 [4.27 pg/ml (2.46-5.99) vs 3.12 pg/ml (1.35-5.03), P=0.036]. Furthermore, smokers had higher serum levels of IL6 compared to ex-smokers [10.19 pg/ml (4.92-17.38) vs 4.46 pg/ml (1.88-11.53), P=0.009]. Finally, lower serum levels of IL6 &lt; median value (9.55pg/ml) were associated with improved survival (median overall survival 338 days vs 237 days Log Rank test, P= 0.046). Conclusions: Elevated levels of measured cytokines were found in both EBC and serum of lung cancer patients. Particularly, IL6 levels were related to T-stage and smoking status, while lower serum levels of IL6 with survival. These findings suggest that cytokines should be further studied in order to evaluate their diagnostic and prognostic significance. EBC could provide a simple, noninvasive and economic method for the monitoring of biomarkers in patients with lung cancer.