Semi-quantitative ELISA Research Articles

Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes

BackgroundNeuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1α and HIF-1β subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1β in astrocytes is mediated by HIF-1α. In this study, we demonstrate the role of HIF-1α in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively.MethodsPrimary fetal human astrocytes or mouse astrocytes generated from HIF-1α+/+ and HIF-1α+/- mice were subjected to hypoxia (<2% oxygen) or 125 μM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1α, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1α with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1α was analyzed by reporter gene assay.ResultsSequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1α inducer, CoCl2, strongly up-regulated HIF-1α expression in astrocytes. Mouse HIF-1α+/- astrocytes had lower basal levels of HIF-1α and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1α+/+ and HIF-1α+/- astrocytes was correlated with the levels of HIF-1α in cells. Both hypoxia and CoCl2 also up-regulated HIF-1α and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes.ConclusionThese findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1α is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.

Enzyme immunoassay for semicarbazide—The nitrofuran metabolite and food contaminant

Semicarbazide (SEM), the marker residue for the banned nitrofuran veterinary antibiotic nitrofurazone (NFZ), has been detected regularly in foods (47% of recent nitrofuran EU Rapid Alerts involve SEM). However, the validity of SEM as a definitive marker for NFZ has been undermined by SEM arising from other sources including azodicarbonamide, a plastics blowing agent and flour treatment additive. An inexpensive screening test for SEM in food matrices is needed—all SEM testing currently uses expensive LC–MS/MS instrumentation. We now report the first production of antibodies against derivatised SEM. A novel carboxyphenyl SEM derivative was used to raise a polyclonal antibody that has been incorporated into a semi-quantitative microtitre plate ELISA, validated according to the criteria set out in Commission Decision 2002/657/EC, for use with chicken muscle. The antibody is highly specific for derivatised SEM, cross-reactivity being 1.7% with NFZ and negligible with a wide range of other nitrofurans and poultry drugs. Samples are derivatised with o-nitrobenzaldehyde and simultaneously protease digested before extraction by cation exchange SPE. The ELISA has a SEM detection capability (CC β ) of 0.25 μg kg −1 when a threshold of 0.21 μg kg −1 is applied to the selection of samples for confirmation (lowest observed 0.25 μg kg −1 fortified sample, n = 20), thus satisfying the EU nitrofurans’ minimum required performance limit of 1 μg kg −1. NFZ-incurred muscles (12) containing SEM at 0.5–5.0 μg kg −1 by LC–MS/MS, all screened positive by this ELISA protocol which is also applicable to egg and chicken liver.

Impact of Treatment with Infliximab on Anticyclic Citrullinated Peptide Antibody and Rheumatoid Factor in Patients with Rheumatoid Arthritis

To investigate the impact of infliximab treatment on anticyclic citrullinated peptide (anti-CCP) antibody and rheumatoid factor (RF) levels in patients with rheumatoid arthritis (RA). Sera from 33 RA patients receiving infliximab and disease modifying antirheumatic drugs were tested for anti-CCP antibody, IgA-, IgG- and IgM-RF using a commercially available semiquantitative ELISA at baseline, 30 and 54 weeks after treatment. The serum levels of anti-CCP antibody and IgA-RF decreased significantly after 30 weeks (P = 0.002 and 0.024); however, the decrease was not significant at week 54 (P = 0.147 and 0.207). The decrease in IgG-RF level was not significant at 30 and 54 weeks (P = 0.059 and 0.097). IgM-RF levels, however decreased significantly at 30 and 54 weeks (P = 0.002 and 0.004). A strong correlation between anti-CCP and IgA-, IgG- and IgM-RF was observed at baseline (r(s) = 0.48, 0.43, 0.65, P = < 0.05) and after infliximab treatment at 30 (r(s) = 0.45, 0.46, 0.62, P = < 0.05) and 54 (r(s) = 0.49, 0.45, 0.60, P = < 0.05) weeks. Treatment with infliximab results in decreased anti-CCP antibody and IgA-RF early in the course of therapy that is not sustained. IgM-RF declines and remains decreased for at least 54 weeks. Investigations in larger cohorts of RA patients (especially early RA) with longer follow-up are needed to assess the impact of specific therapeutic interventions on anti-CCP antibody and RF levels and the relationship of their levels to disease activity.

The Major Allergen of Olive Pollen Ole e 1 Is a Diagnostic Marker for Sensitization to Oleaceae

Background: Trees of the family Oleaceae are important allergen sources, with a strongly varying geographic distribution. For example, olive pollen is an important allergen source in Mediterranean countries, whereas ash pollen dominates in Northern and Central Europe and North America. The aim of this study was to compare the profiles of olive and ash pollen allergens and to study the degree of cross-reactivity using populations of allergic patients selectively exposed to olive or ash pollen. Methods: Olive and ash pollen extracts were analyzed by IgE immunoblotting using sera from Spanish patients highly exposed to olive pollen and Austrian patients without olive but ash pollen exposure. IgE cross-reactivity was studied by qualitative immunoblot inhibition assays and semiquantitative ELISA inhibitions using olive, ash, birch, mugwort, timothy grass pollen extracts and the major olive pollen allergen, Ole e 1. Results: Spanish and Austrian patients exhibited an almost identical IgE-binding profile to olive and ash pollen allergens, with major reactivity directed against Ole e 1, and its homologous ash counterpart, Fra e 1. IgE inhibition experiments demonstrated extensive cross-reactivity between olive and ash pollen allergens. However, whereas cross-reactions between profilins and calcium-binding allergens also occurred between unrelated plant species, cross-reactivity to Ole e 1 was confined to plants belonging to the Oleaceae. Conclusions: Ole e 1 is a marker allergen for the diagnosis of olive and ash pollen allergy.

Role of IL-12B Promoter Polymorphism in Adamantiades–Behcet's Disease Susceptibility: An Involvement of Th1 Immunoreactivity against Streptococcus Sanguinis Antigen

Adamantiades-Behcet's disease (ABD) is a chronic inflammatory multisystem disorder. Although the precise etiology is unclear, high prevalence of human leukocyte antigen (HLA)-B51 predisposition and predominantly involved T-helper type 1 cells (Th1)-type proinflammatory cytokines and extrinsic Streptococcal infection suggest a substantial association with an immunogenetic basis and strengthens the hypothesis that IL-12, a potent inducer of Th-1 immune reaction, is a putative candidate in its pathogenesis. These clinicopathological findings led us to examine interleukin 12 p40 (IL-12B) promoter polymorphism, for which the 4-base pair (bp) heterozygous insertion has been shown to affect the gene transcription and subsequent protein production. We analyzed IL-12B promoter genotypes in 194 Japanese subjects (92 with ABD and 102 normal controls) by PCR-based restriction enzyme digestion. The frequency of the insertion heterozygosity was significantly higher in patients than in controls (49/92, 53.3% vs 39/102, 38.2%, respectively). Comparing these with HLA haplotype data, this trend was more significant in HLA-B51-negative patients (29/42, 69.0% vs 20/50, 40.0%; P = 0.005). As assessed by semiquantitative reverse transcription-PCR and ELISA, stimulation with Streptococcal antigens specifically increased expression of IL-12 p40 mRNA and protein, in conjunction with IL-12 p70 induction, in peripheral blood mononuclear cells from heterozygous patients. Our results provide evidence for anti-bacterial host response toward Th1-immunity mediated by IL-12 in patients with ABD, and the possible insight into the genetic susceptibility that is independent of HLA background.

Neosporacaninum immunoblotting improves serodiagnosisof bovine neosporosis

Neospora caninum ranges among the major causes of infectious abortion in cattle worldwide. The present study was designed to improve the serodiagnostic tools by complementing a conventional ELISA with a highly sensitive and species-specific N. caninum immunoblot. To evaluate this test combination, sera from several groups of cows were tested. The first group, consisting of experimentally infected calves, showed that immunoblot antibody reactivities were detectable 1 to 3 days earlier than those found in ELISA. The first immunodominant bands that appeared were a 29-kDa (NcSAG1) and a 36-kDa (NcSRS2) antigen. Other groups, based upon naturally infected cattle, were used to compare the diagnostic sensitivity of ELISA and immunoblotting. Overall, N. caninum immunoblotting exhibited a higher sensitivity (98%) than ELISA (87%). Conversely, immunoblotting also confirm in two other cases, true transient negativation in some animals. In general, banding patterns and band staining intensity correlated to the semiquantitative ELISA findings. On the other hand, the banding pattern could not be used to discriminate between sera from animals with a recent abortion and those of cows with latent N. caninum infection. We also addressed putative cross-reactions due to infection with Toxoplasma gondii. Sera from animals with a serologically proven T. gondii infection were either clearly negative by Neospora immunoblotting or they yielded a specific immunoblot antibody profile indicating a double infection with N. caninum. Sera from animals with positive findings in both Toxoplasma and Neospora ELISA thus provided dichotomic results in the immunoblot by allowing to confirm or to rule out the specificity of the antibody reaction in Neospora ELISA. Altogether, our findings demonstrate that N. caninum immunoblotting is a very sensitive and specific complementary tool to improve the serology for N. caninum infections in cattle.

Fibroblast Growth Factor 2 and Transforming Growth Factor β1 Synergism in Human Bronchial Smooth Muscle Cell Proliferation

Bronchial smooth muscle cell (BSMC) hyperplasia is a typical feature of airway remodeling and contributes to airway obstruction and hyperresponsiveness in asthma. Fibroblast growth factor 2 (FGF-2) and transforming growth factor beta1 (TGF-beta1) are sequentially upregulated in asthmatic airways after allergic challenge. Whereas FGF-2 induces BSMC proliferation, the mitogenic effect of TGF-beta1 remains controversial, and the effect of sequential FGF-2 and TGF-beta1 co-stimulation on BSMC proliferation is unknown. This study aimed to assess the individual and sequential cooperative effects of FGF-2 and TGF-beta1 on human BSMC proliferation and define the underlying mechanisms. Mitogenic response was measured using crystal violet staining and [3H]-thymidine incorporation. Steady-state mRNA and protein levels were measured by semiquantitative RT-PCR, Western blot, and ELISA, respectively. TGF-beta1 (0.1-20 ng/ml) alone had no effect on BSMC proliferation, but increased the proliferative effect of FGF-2 (2 ng/ml) in a concentration-dependent manner (up to 6-fold). Two distinct platelet-derived growth factor receptor (PDGFR) inhibitors, AG1296 and Inhibitor III, as well as a neutralizing Ab against PDGFRalpha, partially blocked the synergism between these two growth factors. In this regard, TGF-beta1 increased PDGF-A and PDGF-C mRNA expression as well as PDGF-AA protein expression. Moreover, FGF-2 pretreatment increased the mRNA and protein expression of PDGFRalpha and the proliferative effect of exogenous PDGF-AA (140%). Our data suggest that FGF-2 and TGF-beta1 synergize in BSMC proliferation and that this synergism is partially mediated by a PDGF loop, where FGF-2 and TGF-beta1 upregulate the receptor (PDGFRalpha) and the ligands (PDGF-AA and PDGF-CC), respectively. This powerful synergistic effect may thus contribute to the hyperplastic phenotype of BSMC in remodeled asthmatic airways.

Proteomic-Based Discovery and Characterization of Glycosylated Eosinophil-Derived Neurotoxin and COOH-Terminal Osteopontin Fragments for Ovarian Cancer in Urine

The objective was to identify and characterize low molecular weight proteins/peptides in urine and their posttranslational modifications that might be used as a screening tool for ovarian cancer. Urine samples collected preoperatively from postmenopausal women with ovarian cancer and benign conditions and from nonsurgical controls were analyzed by surface-enhanced laser desorption/ionization mass spectrometry and two-dimensional gel electrophoresis. Selected proteins from mass profiles were purified by chromatography and followed by liquid chromatography-tandem mass spectrometry sequence analysis. Specific antibodies were generated for further characterization, including immunoprecipitation and glycosylation. Quantitative and semiquantitative ELISAs were developed for preliminary validation in patients of 128 ovarian cancer, 52 benign conditions, 44 other cancers, and 188 healthy controls. A protein (m/z approximately 17,400) with higher peak intensities in cancer patients than in benign conditions and controls was identified and subsequently defined as eosinophil-derived neurotoxin (EDN). A glycosylated form of EDN was specifically elevated in ovarian cancer patients. A cluster of COOH-terminal osteopontin was identified from two-dimensional gels of urine from cancer patients. Modified forms EDN and osteopontin fragments were elevated in early-stage ovarian cancers and a combination of both resulted to 93% specificity and 72% sensitivity. Specific elevated posttranslationally modified urinary EDN and osteopontin COOH-terminal fragments in ovarian cancer might lead to potential noninvasive screening tests for early diagnosis. Urine with less complexity than serum and relatively high thermodynamic stability of peptides or metabolites is a promising study medium for discovery of the novel biomarkers which may present in many non-urinary tract neoplastic diseases.

β-Casomorphin-7 regulates the secretion and expression of gastrointestinal mucins through a μ-opioid pathway

We have recently shown that beta-casomorphin-7, a milk opioid peptide, strongly stimulates mucin secretion in the rat jejunum through a nervous pathway and opioid receptor activation. In this study, the hypothesis that beta-casomorphin-7 may also act directly on intestinal goblet cells was investigated in vitro in rat and human intestinal mucin-producing cells (DHE and HT29-MTX) using quantitative and semiquantitative RT-PCR and ELISA. The presence of mu-opioid receptors was demonstrated in rat goblet cells in the upper half of the colonic crypt and in the two cell lines by immunohistochemistry and RT-PCR. In rat DHE cells, beta-casomorphin-7 increased the expression of rat mucin (rMuc)2 and rMuc3 but not rMuc1, rMuc4, and rMuc5AC. This effect was time and dose dependent, with the maximum of increase in transcripts being noticed for a concentration of 10(-4) M after 2 h of stimulation for rMuc2 (225% of controls) and 4 h of stimulation for rMuc3 (208% of controls). Mucin secretion was maximally increased after 8 h of stimulation. Interestingly, these effects were prevented by pretreatment of the cells with the mu-opioid antagonist cyprodime. In human HT29-MTX cells, beta-casomorphin-7 (10(-4) M) also increased MUC5AC mRNA levels (219% after 24 h of stimulation) and the secretion of this mucin (169% of controls). In conclusion, beta-casomorphin-7 may contribute significantly to mucin production via a direct effect on intestinal goblet cells and the activation of mu-opioid receptors. Because intestinal mucins have a crucial mucosal protective function, dairy products containing beta-casomorphin-7 may improve intestinal protection and could have dietary and health applications.

Analysis of Accumulation Patterns of Barley yellow dwarf virus-PAV (BYDV-PAV) in Two Resistant Wheat Lines

Barley yellow dwarf (BYD) is one of the main viral diseases of small-grain cereals. This disease, reported on numerous plant species of the Poaceae family, is caused by a complex of eight viral species including the species Barley Yellow Dwarf Virus-PAV (BYDV-PAV), frequently found in western Europe. Resistance sources against BYDV-PAV are scarce and only identified in perennial Triticineae. Some BYDV-resistant wheat lines have been obtained by introgressing these resistances into bread wheat germplasms. Genetic and biological characterization of the resulting lines has been undertaken. However, little information on the resistant behaviour of these lines during the early stages of the infection process is available. To evaluate the resistance of two genetically distinct resistant lines (Zhong ZH and TC14), 1740 young plantlets, belonging to susceptible reference hosts (barley cv. Express and wheat cv. Sunstar), Zhong ZH or TC14 wheat lines, were inoculated in controlled conditions with French BYDV-PAV isolates. The infection process was monitored during the first 21 days after inoculation (DAI) using a semi-quantitative ELISA. A standardized protocol including five successive samplings of leaves from all inoculated plants and the collection of plant roots at the end of the monitored period was carried out. This protocol enabled an assessment of the infection percentage and the evolution of the viral load in plants from the 7th DAI to the 21st DAI. Statistical analyses of the BYDV infection kinetics using raw ELISA data, a model of the time-dependent variation of the percentage of infected plants and the area under concentration progress curves (AUCPC) demonstrated that Zhong ZH and TC14 lines (1) reduce the development rate of the BYD disease during the first days of infection, (2) decrease the infection efficiency of BYDV-PAV isolates, in the leaves, from 98.7% for susceptible plant genotypes to 81.9% and 71.7% for Zhong ZH and TC14, respectively, (3) reduce the virus load in the leaves of infected plants and (4) are not spared from BYDV infection, as 95.1% of Zhong ZH and 90.2% of TC14 inoculated plants accumulated viral particles in roots and/or in leaves at 21 DAI. These results confirm the BYDV-partial resistant behaviour of both Zhong ZH and TC14 lines. The development rate of the disease was the single parameter that allowed the distinction between the two resistant sources present in the tested lines.

Semiquantitative Detection of Abnormal CD44 Transcripts in Colon Carcinomas by Reverse Transcription-Polymerase Chain Reaction Enzyme-linked Immunosorbant Assay (RT-PCR ELISA).

Background: Abnormal expression of CD44 variant RNA has been detected in a variety of human tumors and has been shown to be a potential diagnostic marker. To date, such analysis requires time-consuming gel electrophoresis, blotting, and autoradiographic procedures, and this approach may not be suitable for routine laboratory examinations. We have developed a rapid and semiquantitative reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay (RT-PCR ELISA) method and used it to analyze CD44 expression in colon carcinoma tissues and exfoliated cancer cells in colon luminal washings. Methods and Results: RNA was extracted from sample cells and tissues and converted to cDNA. PCR amplification products, labeled by incorporation of digoxigenin-11-dUTP, were hybridized with biotinylated probes complementary to CD44 exon 12 or to exons in the standard portion (CD44s) of the gene. Hybridized DNA complexes were immobilized on streptavidin-coated microtiter plates, and the bound PCR products were detected with a peroxidase-conjugated antibody to digoxigenin. CD44-derived PCR products were quantified by absorbance of a chromogenic reaction. Elevated expression of CD44 variant exon 12 was detected initially by Southern blot analysis in all of the 9 colon carcinoma tissues, while weak expression was observed in only 3 of 9 normal mucosas. This tumor-related differential expression was confirmed by the newly developed PCR-ELISA method. Elevated expression of CD44 exon 12 was also detected in exfoliated colonic epithelial cells from 10 of 13 carcinoma cases but not in exfoliated cells from 4 patients with inflammatory bowel disease. Conclusions: Raised expression of CD44 variant exon transcripts can be detected reliably in colonic tumor tissue and in exfoliated colonic cancer cells by a semiquantitative RT-PCR ELISA method. This was shown to be as sensitive as conventional RT-PCR using chemiluminescent detection. Therefore, CD44-based RT-PCR ELISA could facilitate detection of neoplasia in clinical specimens including colon washings and naturally micturated urine.

Donor-Derived Hematopoietic Stem Cells Repair the Bone Marrow Sinusoids Following Bone Marrow Transplant.

Marrow sinusoidal capillaries provide a niche for megakaryocyte progenitors and possibly hematopoietic stem cells. We sought to determine the fate of host sinusoidal capillaries during marrow transplant. We transplanted whole bone marrow, 2000 sorted Linneg Sca-1pos c-kitpos (SKL) hematopoietic stem and progenitor cells, or single SKL cells (along with rescue marrow) from male mice expressing green fluorescent protein into lethally irradiated female C57/BL6 hosts. We used green fluorescence and the presence of the Y-chromosome to identify donor-derived cells. Sinusoidal engraftment was studied from day 5 up to 1 year post-transplant and in secondary transplants. We identified numerous donor-derived cells based on green fluorescence or presence of the Y chromosome lining the sinusoids, but it was unclear whether these cells were vascular or hematopoietic. Furthermore, we were unable to demonstrate expression of von Willebrand factor, CD31 and MECA-32 using immunohistochemistry in healthy sinusoids. We used two methods to definitively identify donor-derived endothelial cells. First, we identified these cells based on location, characteristic shape and nuclei on H&E stained sections. Serial high power fields were photographed and then the same sections were stained with X and Y FISH probes. Second, we identified donor endothelial cells based on uptake of intravenously injected Ac-LDL which was endocytosed predominantly by sinusoidal endothelial cells four hours after injection. Flushed femoral bone marrow was treated with dispase and collagenase, and disaggregated cells were lineage depleted with a standard lineage cocktail containing Mac-1 antibody. Cytospins were photographed, Ac-LDL staining cells identified, and the same cytospins were stained with X and Y FISH probes. We counted numerous donor-derived endothelial cells whether 2X106 whole bone marrow (n=6 ), 2X104 SKL cells (n=6), or single SKL cells (n=2) were transplanted. These donor-derived endothelial cells were functional based on their ability to uptake DiI AcLDL. These cells appeared adjacent to mature, donor-derived megakaryocytes, suggesting function as a niche for megakaryocyte progenitors. Furthermore, donor-derived endothelial cells were present at levels similar to hematopoietic engraftment in every animal analyzed, suggesting robust levels of repair. Single cell transplants were assessed at seven and nine months, and in secondary transplants, establishing that these cells self-renew. To determine the mechanism whereby HSC's repair the bone marrow sinusoids, we measured the expression of stromal derived factor-1 (SDF-1) at various time points during the first two weeks post-transplant by semi-quantitative RT-PCR, ELISA, and immunohistochemistry. SDF-1 expression peaked 3 days post-transplant and was expressed primarily by damaged blood vessels. The temporal and spatial pattern of engraftment matched expression patterns of SDF-1. This study demonstrates repair of host sinusoids by donor-derived endothelial cells following transplant, with levels of reconstitution similar to that of the hematopoietic system. This repair may be mediated by expression of SDF-1 by damaged marrow vasculature. Repair of sinusoidal capillaries may be a primary role of the HSC during transplant that is necessary for successful hematopoietic engraftment.

Analysis of myosin isoform transitions during growth and development in diverse chicken genotypes

The temporal expression of chicken skeletal fast myosin heavy chain (MyHC) isoforms in pectoralis major muscle was characterized in 3 commercial broiler lines at embryonic d 19 and at 7, 14, and 21 d posthatch. Lines A and B have been selected for breast yield, and line C is a fast' growing commercial line with limited selection for carcass traits. The isoform transitions in breast muscle samples were compared with samples from Single Comb White Leghorns (line D) using a semiquantitative immunoassay. The hypothesis was that selection for growth and carcass development in broilers would be accompanied by changes in the temporal expression of one or more of the chicken fast MyHC isoforms. Embryos from all lines were sampled at 19 d of incubation, and chicks were randomly sampled at 7, 14, and 21 d post-hatch. Myosin was extracted from pectoralis major muscle and assayed for purity and total protein concentration by SDS-PAGE and bincinchoninic acid protein analyses, respectively. The relative concentration of MyHC isoforms was evaluated by semiquantitative ELISA with 3 monoclonal antibodies specific for chicken skeletal fast embryonic and adult (eMyHC, aMyHC; EB165), neonatal (nMyHC; 2E9), and adult (aMyHC; AB8) myosin, respectively. The overall temporal expression of the myosin isoforms, eMyHC, nMyHC, and aMyHC, was similar in all lines. With eMyHC, at 19 d of incubation, line B had lower expression than lines A, C, and D. Expression of nMyHC, in lines C and D was similar with expression being highest at 7 d and lower at 14 d and 21 d. In lines A and B, however, nMyHC expression was higher at hatch than lines C and D. In line D, aMyHC was expressed at 14 d and increased through 21 d, whereas in lines A, B, and C, aMyHC isoform was expressed and was higher at 7 d and increased through 21 d. The results of this experiment support our hypothesis that commercial broilers have different temporal expression patterns of the developmental chicken fast MyHC isoforms.

Phenytoin can accelerate the healing process after experimental myocardial infarction?

Background Over-degradation and/or inadequate accumulation of extracellular matrix after myocardial infarction (MI) may lead to adverse ventricular remodeling, even ventricular aneurysm or rupture. Phenytoin can increase gingival overgrowth by stimulating the proliferation of connective tissue, which implies a novel way to hasten the healing process after MI. Methods Experimental MI was induced by permanent coronary ligation. Surviving rats after MI were randomly divided into phenytoin, captopril, phenytoin plus captopril, operation control and sham operation group. Picrosirius red staining plus polarized microscopy was used for collagen analysis. Left ventricular passive pressure–volume relationship was determined ex vivo. The effects of phenytoin concentration gradient (0, 1.25, 2.5, 5.0, 10.0, and 20.0 μg/mL) on transforming growth factor-β 1 (TGF-β 1) mRNA and protein expression by neonatal rat cardiac fibroblast were determined using semi-quantitative RT-PCR and ELISA, respectively. Peritoneal macrophage was incubated with same gradient of phenytoin concentration. Then the supernatant was harvested to stimulate another 6 groups of cardiac fibroblast, to investigate possible role mediated by macrophage. Results Phenytoin treatment could promote type I collagen cross-linking level and ratio of type I/III collagen in the infarcted region and had no obvious side effect on interstitial collagen volume fraction, subtype ratio and distribution in non-infarcted region. Phenytoin-treated hearts exhibited attenuation of global ventricular dilation. Phenytoin alone had no direct effects on rat cardiac fibroblast proliferation and collagen production in vitro, but phenytoin-stimulated macrophage could exert a positive influence on cardiac fibroblast TGF-β 1 mRNA and protein production, which exhibited a dose-dependent manner. Conclusions Phenytoin can accelerate the healing process in the infarcted region and has no obviously detrimental influence on collagen accumulation in non-infarcted region, which implies a potential benefit to patients undergoing early post-infarction ventricular remodeling process.

Further studies on hepatitis C virus NS5A–SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.

CD47 Engagement Impairs the Function and Maturation of Human Dendritic Cells Via the Inhibition of NF-κB Avtivity.

OBJECTIVE Dendritic cells (DCs) are important functional antigen-presenting cells that play an essential role in initiating and modulating immune responses. Their mature states are crucial for the induction of T-cell-mediated immune reactions. Therefore, The mechanism of T cell anergy related with DCs and research in applying immature DCs to establish donor-specific immune tolerance have become one of the new frontiers in the field of transplantation immunity. The activation of nuclear factor kappa B(NF-κB) has been said to modulate the maturation of DCs, which controls the transcription of genes encoding major histocompatibility complex(MHC)antigens, and costimulator molecules for T cell activation. The CD47 Ag, a multispan transmembrane ptotein expressed on all hemopoietic cells, has an immunosuppressive function with its ligand, monoclonal antibody (mAb) or thrombospondin. In the current study, we investigate the influence of CD47 engagement by its soluble mAb B6H12 on the maturation and function of cultured DCs. Further to observe any results could be ascribed to the effect of B6H12 mAb on the nuclear translocation and DNA binding of NF-κB.METHODS Monocyte-derived DCs were propagated in granulocyte-macrophage colony stimulating factor (GM-CSF) combined with lipopolysaccharide (LPS) and interleukin(IL)-4, in the presence or absence of soluble anti-CD47 monoclonal antibodies (anti-CD47 mAbs, B6H12). The phenotype of DCs(CD80, CD86, CD83, CD1a, HLA-DR) were detected by the flow cytometry. Semi-quantitative RT-PCR and ELISA methods were used to analyse the mRNA and protein expression levels of interleukin-12(IL-12). The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by Brdu-ELISA. Electrophoretic mobility shift assay (EMSA) was applied to examine the activity of NF-κB.RESULTS Both the immature and the mature DCs express CD47, which has a ratio of 94-98%. The cell immune phenotype was lower in B6H12 mAb treated DC group than that in untreated groups. The data were as follow:CD80+(68.14±7.41)% vs (89.17±8.59)%; CD86+ (67.33±4.71)% vs (87.27±3.56)%;CD83(40.08±14.80)% vs (72.77±8.68)%;Cdla+(66.45±4.06)% vs (95.93±3.03)%; HLA-DR(40.67±13.48) vs (98.97±1.01)%, respectively. The mRNA levels of IL-12 P40 was decreased (P<0.01) with the treatment of B6H12 mAb. Pre-exposure of developing DCs to 10μg/ml B6H12 considerably reduced their subsequent ability to secret IL-12 P70 upon stimulation by LPS, comparing with the other two control groups [(36.6±3.83)pg/ml vs (80.43±8.24)pg/ml vs(83.7±9.58 )pg/ml] (P<0.05). It is also noted that the inhibition of IL-12 P70 release by B6H12 mAb was dependent on dose at the range of 2.5 - 10μg/ml, and the inhibition can be seen in DCs treated with B6H12 mAb at the dose of 2.5μg/ml. The data of the mixed leukocyte reaction were consistent with the flow cytometry results (P<0.01). Pre-exposure to B6H12 mAb during the development of DCs resulted in a dramatic decrease of the DNA binding activity toward protein in the nucleus. Moreover, this inhibition was also seemed to be dose dependent (2.5μg/ml, 5μg/ml, 10μg/ml) and the density scan values were:126.1±4.32, 103.46±10.96, 72.80±10.08.CONCLUSION This results indicate that the CD47 ligation by B6H12 mAb exerts a negative effect on the maturation and function of in vitro cultured DCs through the inhibition of NF-κB binding activity, which suggest its favorable role in the induction of modulation of immune tolerance.

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Endothelial Tissue Factor Pathway Inhibitor.

Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.

P0432 CAN TTG REPLACE SMALL BOWEL BIOPSY TO DIAGNOSE CELIAC DISEASE IN SELECT PEDIATRIC POPULATIONS?

Introduction: The increasingly widespread use of screening tests for celiac disease (CD) has raised the awareness and the number of patients referred for investigation. The current gold standard for the diagnosis of CD is the histological analysis of a small intestinal (SI) biopsy. Commercially available IgA anti-tissue transglutaminase antibody (TTG) screening tests have high sensitivities and specificities using manufacturer recommended cut-off values. We propose that the subgroup of patients with very high TTG titres is positive for CD, a SI biopsy is not always necessary to make the diagnosis and a gluten-free diet should be trialed. If the patient’s symptoms do not improve then a SI biopsy should be performed. Methods: We conducted a retrospective chart review of 104 patients that had both a TTG and SI biopsy at BC Children’s Hospital between September 2000 and October 2003. The biopsies were reviewed by a pathologist blinded to the TTG results and previous biopsy reports. Manufacturer’s recommended and previously validated in hospital TTG value cut-offs were; <20 negative and >=20 positive. Our lab reports individual values from 1 to 100 and >100 using a semi-quantitative ELISA. We used TTG >100 and <20 for cut-offs and suggested biopsying patients with TTG values between 20–100. Results: 59/104 patients were biopsy positive, 14 males and 45 females. 45/104 were negative, 24 males and 21 females. Predispositions to celiac disease included IDDM=14, thyroid disease=4, Down’s syndrome=4, celiac disease in a first degree relative=6 and autoimmune disease in the family=12. Three patients with TTG <20 had positive biopsies, 2 were IgA deficient and 1 had no IgA reported. Predisposition to CD was not related to a TTG >100 or a positive biopsy.Table 1: TTG ValuesConclusion: By changing the cut-off values for our TTG kit to >100 and <20 and checking for selective IgA deficiency, we found TTG to be highly accurate for celiac disease. If patients do not improve on the gluten-free diet, then they should be investigated with a SI biopsy. Patients with TTG values of 20–100 or <20 with known IgA deficiency need to be biopsied to diagnose CD. This proposal could shorten the time to diagnosis, eliminate the need for some patients to undergo a SI biopsy and save patient and hospital expenses.

Detection of central nervous system tissues as BSE specified risk material in meat products in Turkey

Abstract Spinal cord and brain tissues have the highest importance to transfer bovine spongiform encephalopathy (BSE) to human. European Union issued complete removal of spinal cord and brain tissues from meat products (Commission Decision 2000/418/EC). In Turkey, regulation on meat products [Turkish Food Code, Regulation on Meat Products. Official Journal No: 23960, 10 Feb, 2000] is applied by their government veterinarians. But there are some questions on the effective application of the regulation especially in some private slaughterhouses. This study was designed to represent the mixing of these tissues to meat products which sold in Marmara region of Turkey. For this purpose, a total of 179 meat product samples belonged to 17 different trademarks were tested. A semi-quantitative ELISA kit, which detects glial fibrillary acidic protein (GFAP) as marker, was used. In the test, standard controls 0, 0.2%, 1%, and 2% were practiced. Of the 179 sampled, 18 samples (10.05%) gave an optical density over than standard 0.2%. Optical density of three samples was near to standard 1%. It is concluded that central nervous system tissue mixing to the meat products still happen and more effective control measurements needs to be developed and applied in Turkey.

Endothelin-1 down-regulates the expression of vascular endothelial growth factor-A associated with osteoprogenitor proliferation and differentiation

Endothelin-1 (ET-1) is implicated in the signaling between vascular endothelial cells (VECs) and osteoblasts during bone development, remodeling and repair. Vascular endothelial growth factor (VEGF) also plays an important role in these intercellular interactions. Our objectives were to identify which specific VEGF isoforms were produced during osteoblastic proliferation and differentiation and to determine the effects of ET-1 on VEGF mRNA and protein production by osteoblastic cells. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and ELISA were used to evaluate VEGF mRNA isoform expression and protein synthesis at different stages of ET-1-induced osteoblastic differentiation in fetal rat calvaria (FRC) osteoblastic cells. Three VEGF mRNA isoforms were identified corresponding to VEGF 120, VEGF 164 and VEGF 188. Predominant isoforms VEGF 120 and VEGF 164 had a bimodal expression that increased in the early proliferation and late mineralization phases. ET-1 stimulated osteoblastic proliferation and differentiation, but surprisingly, ET-1 down-regulated VEGF mRNA and protein expression and sustained the down-regulation over time in long-term cultures. Time course studies showed that ET-1 inhibited VEGF mRNA expression after incubation for 3 h in 7- and 14-day FRC cell cultures. Similarly, ET-1 inhibited VEGF protein secretion by 5.8- and 2.8-fold in 7- and 14-day FRC cells, respectively. VEGF-A protein secretion was inhibited by ET-1 in a dose-dependent manner with a maximal effect at 10 −7 M. This study supports a novel inhibitory role for ET-1 on VEGF synthesis in osteoblastic cells as a feedback mechanism in the temporal and spatial coupling of angiogenesis to bone formation and resorption.