bluuebunny/crossref_metadata_2025 huggingface dataset:
Digoxin-like immunoreactivity in normal human plasma and urine, as detected by a solid-phase radioimmunoassay.
Authors: Balzan S , Clerico A , Grazia del Chicca M , Montali U , Ghione S
Published: January 2020

Reports on the presence of digoxin-like immunoreactive substance(s) (DLIS) in the plasma and urine of several animal species and a few human test subjects prompted us to undertake to confirm the presence of DLIS in plasma and urine of normal persons and to investigate some characteristics of this antibody-DLIS binding. For this purpose we used a modified radioimmunoassay kit involving antidigoxin antibody-coated test tubes and 125I-labeled digoxin to measure DLIS in urine and concentrated plasma of ostensibly healthy subjects. In 12 separate experiments with plasma the mean sensitivity was 5.12 (SD 1.11) pg per tube, expressed as digoxin. There was no significant cross reactivity with human serum albumin in concentrations up to 200 g/L. The mean DLIS value (in digoxin equivalents) for plasma from 24 normal subjects was 33.58 (SD 14.24) ng/L. Its mean concentration in urine from five subjects was 315.00 (SD 91.38) ng/L.
Tartrate-resistant acid phosphatase in serum of cancer patients.
Authors: Lam K W , Dannaher C , Letchford S , Eastlund T , Li C Y , Yam L T
Published: January 2020

Tartrate-resistant acid phosphatase activity determined by enzyme immunoassay was higher in the serum of cancer patients than that in normal blood donors. The highest activity was found among patients having malignancy metastatic to bone. The classic colorimetric method showed a broad range of values among normal blood donors, and the contrast between normal and cancer patients was less obvious. Most of the cancer patients had normal to low alkaline phosphatase activities.
Selenium and other elements in human maternal and umbilical serum, as determined simultaneously by proton-induced X-ray emission.
Authors: Hyvönen-Dabek M , Nikkinen-Vilkki P , Dabek J T
Published: January 2020

Using PIXE (proton-induced X-ray emission), we simultaneously determined the concentrations of Se, Ca, Fe, Cu, Zn, Br, and Pb in blood serum from 56 pregnant women, 25 healthy controls, and 31 others with twin pregnancy or some complicating condition (diabetes, hypertension, epilepsy, hepatosis gravidarum, pre-eclampsia, small baby), and in cord-blood serum from 21 newborns. Pellets, pressed from the serum samples after addition of yttrium as an internal standard, mixing, and evaporating at 30 degrees C with or without reduced pressure (less than 1 kPa), were bombarded by 2.2 MeV protons from a Van de Graaff accelerator in the air and the induced X-rays collected by a Ge(Li) detector. Relative to mean Se values for early six- to 12-week pregnancy (0.045 ppm), those for 35-42 week pregnancy (0.028 ppm) were low (p less than 0.001). Umbilical cord blood serum showed even lower values (0.016 ppm, p less than 0.001)--findings in harmony with the incidence pattern of Keshan cardiomyopathy. Pb crossed the placenta; values for cord serum were not significantly different from those in pregnancy serum. Cu, Zn, Fe, and Ca showed the significant expected patterns in the different groups. Compared with the late-pregnancy controls, Fe was high in mothers of small-birth-weight babies (1.70 ppm, p less than 0.02). Br was high in pre-eclampsia (3.59 ppm, p less than 0.05) and mothers with twins (3.61 ppm, p less than 0.05).
Interferences with potentiometry of CO2 in the Ektachem 400 Analyzer.
Authors: Steelman M , Smith C H , Menon A , Thach B T , Hillman R E , Landt M
Published: January 2020

We have investigated the Kodak Ektachem 400 Analyzer procedure for CO2 for interferences from benzyl alcohol, benzoic acid, and several compounds structurally similar to benzoic acid. Benzoic acid in plasma, at concentrations found in neonates intoxicated with benzyl alcohol, caused a large increase in the results for CO2, as did substantially above-normal concentrations of certain fatty acids and keto-acids, and toxic concentrations of aspirin. We observed a correlation between increasing benzoic acid concentrations (up to 17 mmol/L) and falsely increasing CO2 values (greater than 47 mmol/L) obtained with the Ektachem Analyzer for samples from a neonate in the intensive-care unit, who was receiving benzyl alcohol-preserved saline solutions. Although the Ektachem CO2 procedure is simple and rapid, and in most cases accurate, questionable results are occasionally encountered, as indicated by a low anion gap or a measured CO2 exceeding that calculated from blood gas measurements. Such results require the use of another method for verification.
Inhibition of cholesterol oxidase in a patient with Cushing's syndrome.
Authors: Davey D D , Alber D B , Ryder K W , Jones J M , Hostetler M L , Moorehead W R , Oei T O
Published: January 2020

Serum from a patient with Cushing's syndrome who was being treated with mitotane contained components that interfered with determination of cholesterol in the Du Pont aca. A measured concentration of cholesterol of 4.19 g/L in the undiluted serum increased to a calculated concentration of 9.50 g/L in diluted serum. Adding additional cholesterol oxidase (EC 1.1.3.6) overcame the reaction inhibition in the undiluted sample; adding additional cholesterol esterase (EC 3.1.1.13) had no effect. There is the potential for clinically significant underestimation of cholesterol with the aca in such patients, because the interference may remain undetected. On the other hand, the aca accurately quantified undiluted specimens containing as much as 10 g of cholesterol per liter when mitotane was not present.
Direct spectrophotometry of serum hemoglobin: an Allen correction compared with a three-wavelength polychromatic analysis.
Authors: Noe D A , Weedn V , Bell W R
Published: January 2020

An Allen correction and a polychromatic analysis are about equally effective in minimizing effects of interference by bilirubin and triglyceride turbidity in the direct spectrophotometry of serum hemoglobin: interference from bilirubin is nearly eliminated, that from turbidity substantially decreased. The limit of detectability of hemoglobin is 8 mg/L in the presence of a moderate concentration of bilirubin. A change in hemoglobin concentration as small as 16 mg/L can be detected in serum having a concentration near the upper limit of the reference interval, i.e., at the medical decision level. The polychromatic formula gives concentration estimates approximately 5% greater than those of the Allen correction. The formula for the Allen correction is hemoglobin (mg/L) = 1.68 mA415 - 0.84 mA380 - 0.84 mA450 . That for the polychromatic analysis is hemoglobin (mg/L) = 1.65 mA415 - 0.93 mA380 - 0.73 mA470 .
Apolipoprotein E polymorphism and hyperlipidemia.
Authors: Assmann G , Schmitz G , Menzel H J , Schulte H
Published: January 2020

We tested apolipoprotein E phenotypes in 1557 normolipidemic factory workers and 822 hyperlipidemic hospital patients. We distinguished six different apolipoprotein E phenotypes and determined their frequencies in normolipidemia (factory workers), hypertriglyceridemia, hypercholesterolemia, and mixed hyperlipidemia. For the three homozygous phenotypes E3/3, E4/4, and E2/2, the percentage distribution in the normolipidemic group was 62.2%, 2.2%, and 0.9%, respectively; for the three heterozygous phenotypes E4/3, E3/2, and E4/2, we determined frequencies of 19.9%, 11.7%, and 2.9%, respectively. A higher prevalence of E2/2 homozygosity was observed in hypertriglyceridemic persons (2.5%) and persons affected by mixed hyperlipidemia (5.0%). E4/4 homozygosity occurred more often among hypercholesterolemic patients (5.0%) than normolipidemic persons (2.2%). These data suggest that E2/2 homozygosity and E4/4 homozygosity both predispose to hyperlipidemia. Patients affected by mixed hyperlipidemia should be investigated for their apolipoprotein E polymorphism because of the possible linkage of apolipoprotein E2/2 homozygosity, hyperlipidemia, and atherosclerosis.
Trace elements in serum from Pakistani patients with acute and chronic ischemic heart disease and hypertension.
Authors: Khan S N , Rahman M A , Samad A
Published: January 2020

We examined sera from 159 patients with ischemic heart disease and hypertension and from 50 apparently healthy control subjects for content of trace elements, cholesterol, triglyceride, and enzymes. Concentrations of copper, cobalt, cholesterol, and triglyceride were increased in all patients, but calcium was decreased in patients with hypertension, acute myocardial ischemia, and acute myocardial infarction. Also accompanying acute myocardial infarction were decreased concentrations of zinc and iron but increases in nickel, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase. Magnesium concentration was lower in patients with acute myocardial ischemia. In acute myocardial infarction, the concentrations of copper, zinc, and iron were higher after 21-30 h (as compared with the values at 0-10 h), by which time concentrations of calcium, magnesium, cobalt, and alanine aminotransferase had decreased. The variation in concentration of trace elements in serum from cases of ischemic heart disease and hypertension corresponds to the severity of the disorder.
Carboxypeptidase-catalyzed hydrolysis of C-terminal lysine: mechanism for in vivo production of multiple forms of creatine kinase in plasma.
Authors: Perryman M B , Knell J D , Roberts R
Published: January 2020

Human myocardial creatine kinase isoenzyme MM is present as a single form in tissue, but upon its release into plasma two additional forms, with faster anodal migration, are apparent on polyacrylamide electrophoresis. We designate the three forms as MM3, MM2, and MM1 in increasing order of anodal mobility. When tissue creatine kinase isoenzyme MM (MM3) is incubated with either carboxypeptidase N or carboxypeptidase B it is converted into the two additional forms, MM2 and MM1. The carboxy terminal amino acid of human, canine, and rabbit tissue MM3 was determined to be lysine, a specific substrate for carboxypeptidases N and B. Evidently the mechanism for the production of multiple forms of creatine MM in human plasma is the hydrolysis of a positively charged C-terminal lysine residue from one M subunit (MM2), followed by hydrolysis of the C-terminal lysine from the other subunit (MM1).
Quantification of pepsin A activity in canine and rat gastric juice with the chromogenic substrate azocoll.
Authors: Will P C , Allbee W E , Witt C G , Bertko R J , Gaginella T S
Published: January 2020

The activity concentration of pepsin may be quantified by using azocoll as a chromogenic substrate. The measured enzyme activity is constant between pH 1.2 and 3.4 and is proportional (r = 0.61) to the activity measured with hemoglobin as substrate. The activity of purified porcine pepsin is inhibited by pepstatin A with an apparent Ki of 115 nmol/L. The azocoll method is useful for measuring changes in pepsin secretion in response to pharmacological agents. For example, pepsin activity of canine gastric juice is decreased by 80% after in vivo administration of 0.5 mg of the synthetic trimethyl prostanoid Ro 22-6923 per kilogram of body weight. The method is sufficiently sensitive to measure the pepsin activity in 0.2 microL of canine gastric juice with a CV of approximately 10%, is simpler than the hemoglobin-substrate methods, and the substrate is commercially available.
Sensitive non-radioisotopic method for measuring lipoprotein lipase and hepatic triglyceride lipase in post-heparin plasma.
Authors: Nozaki S , Kubo M , Matsuzawa Y , Tarui S
Published: January 2020

In this method for measuring lipoprotein lipase (LPL) and hepatic triglyceride lipase (H-TGL) in post-heparin plasma, we determine the released free fatty acids enzymically. After release, they are extracted by Dole 's method (J. Biol. Chem. 235: 2595-2599, 1960), solubilized with Triton X-100, then measured with an enzymic kit (NEFA Kit-K; Nippon Shoji Kaisha Ltd.) after residual turbidity is removed by centrifugation with chloroform. A 5-microL sample of post-heparin plasma suffices to measure the activity of LPL and H-TGL; thus the method is as sensitive as the radioisotopic method. Selective assay of LPL and H-TGL, by adding sodium dodecyl sulfate to inactivate H-TGL or NaCl to inactivate LPL, is also feasible. The mean activities +/- SD of LPL and H-TGL in plasma of normal healthy men were respectively 9.4 +/- 2.3 mumol/h per milliliter (157 +/- 38 U/L) and 20.1 +/- 10.4 (335 +/- 173 U) mumol/h per milliliter (U/L).
A direct comparison of two slope-estimation techniques used in method-comparison studies.
Authors: Parvin C A
Published: January 2020

Least-squares regression estimation of the slope and intercept in method-comparison studies is the most common method of data evaluation despite well known theoretical difficulties with this approach. An alternative estimation technique has been suggested, which avoids some of these theoretical difficulties. I developed an exact mathematical relationship between these two slope estimates in order to directly compare their characteristics. A simple approximate formula for the relative difference between the two slope estimates is also derived. Using these mathematical relationships, I compare the two estimation techniques, using data from published method-comparison studies. In most of the published method comparisons the differences between slope estimates were trivial, but in some the differences were large enough to have warranted use of the alternative slope estimate.
Optimized liquid-chromatographic determination of metronidazole and its metabolites in plasma.
Authors: Gibson R A , Lattanzio L , McGee H
Published: January 2020

Metronidazole and its known metabolites in plasma can be rapidly separated by a "high-pressure" liquid-chromatographic method that can also be adapted for rapid determination of tinidazole. Samples deproteinized with trichloroacetic acid (50 g/L final concentration) undergo isocratic separation on a reversed-phase C18 column eluted with an 8/92 (by vol) mixture of acetonitrile/KH2PO4 (5 mmol/L, pH 3.0). The method is sensitive, reliably detecting as little as 25 micrograms of metronidazole and (or) its metabolites per milliliter of plasma. The detector response varied linearly with concentration for all compounds tested over a wide range (25-500 micrograms/L). Within-day and between-day variation was generally less than 2.5% for all concentrations of all compounds tested. Various other antibiotics tested did not interfere.
Self testing, an emerging component of clinical chemistry.
Authors: Free A H , Free H M
Published: January 2020

Self testing as a phase of health delivery has been utilized for approximately 70 years, but within the last two decades there has been an increasing interest in and use of self testing. The practice of self testing has a sound basis, which involves medical relevancy, cost containment, and convenience. It is reasonable to anticipate that self testing will increase in the future as medical needs and available procedures are established. The clinical chemist, the physician, and the industrial producer of test systems all have important roles in self testing. The clinical chemist, whether in a hospital laboratory or in an industrial setting, has the responsibility to create new self-testing procedures that represent expansion and improvement over those currently available. The clinical chemist also has a critical role in the evaluation of new self-testing procedures and in validating their capability of providing high-quality information. The provision of quality-assurance programs and proficiency programs for the user can most effectively be carried out by clinical chemists. The clinical chemist can also play an important role as a consultant, teacher/educator, and trouble shooter in recognizing and helping solve problems that may appear in various areas of self testing. The classic role of the physician has been to diagnose disorders of the patients and provide therapy for effective care. The role of the physician in self testing is quite comparable to his usual role, since the ultimate diagnosis of disease will be carried out by the physician and he will continue to be the source for definition of therapy. A major portion of self testing will be carried out with the recommendation of a physician, and he will maintain a role in the interpretation of results. The physician needs to be familiar with self-testing practices and procedures and be prepared to provide interpretation of self-testing results. Industry supplies an increasing proportion of reagent systems and instruments for the clinical laboratory. The supplying of reagent systems and small instruments for self testing is almost completely a role of industry. The creation and funding of new products for self testing will be provided by industry to a large extent. A critical function of industry is to provide high-quality products and efficient customer service to the self-testing component of health delivery. We have each played a role in self testing over a period of approximately 40 years. During this period self testing has become an important phase of clinical laboratory practice.(ABSTRACT TRUNCATED AT 400 WORDS)
A specific kinetic assay for tripeptide aminopeptidase in serum.
Authors: Kanda S , Sudo K , Kanno T
Published: January 2020

This is a method for measuring tripeptide aminopeptidase (EC 3.4.11.4) activity in serum. L- Leucylglycylglycine is used as substrate, and the reaction is followed by monitoring the absorbance increase at 340 nm when NAD+ is reduced to NADH in the presence of an excess of leucine dehydrogenase. This principle allows kinetic determination of the enzyme without interference by carboxypeptidases. Amastatin is added to the reaction mixture to prevent nonspecific hydrolysis of the substrate catalyzed by other aminopeptidases. As final reaction concentrations we recommend (per liter): 100 mmol of Tris buffer (pH 8.2), 4.0 mmol of L- leucylglycylglycine , 10 kU of leucine dehydrogenase, 3.8 mmol of NAD+, and 85 mumol of amastatin . The assay is suited to modern enzyme analyzers and has high precision.
Automated kinetic determination of angiotensin-converting enzyme in serum.
Authors: Harjanne A
Published: January 2020

In this automated kinetic modification of a previous method (Anal Biochem 95: 540-548, 1979) for determining angiotensin-converting enzyme (EC 3.4.15.1), 3-(2- furylacryloyl )-L- phenylalanylglycylglycine is used as the substrate. The change in absorbance at 340 nm is used to monitor hydrolysis of the substrate. The rate of hydrolysis is roughly threefold greater than with previously reported substrates, so assay time and sensitivity are improved.
Differential immunoadsorption coupled with rate nephelometry for estimation of DNA-binding immunoglobulins.
Authors: DeBari V A , Nicotra J , Blaney J F , Schultz E F , Needle M A
Published: January 2020

We describe a technique for estimating the mass of anti-DNA antibodies by immunonephelometry of serum immunoglobulins (IgG, IgA, IgM) before and after adsorption onto DNA bound to agarose-polylysine columns. Sixteen patients with systemic lupus erythematosus and 16 age- and sex-matched controls were studied. Precision was determined for high-value (in 10 patients) and low-value (in nine controls) ranges for each of the immunoglobulins. Within-run CVs ranged from 3.0% (IgG, controls) to 11.8% (IgA, patients); between-run CVs ranged from 15.5% (IgG, patients) to 25.2% (IgM, patients). We found anti-DNA antibody concentrations (mean +/- SD) in systemic lupus erythematosus of 1.981 +/- 1.015 g/L for IgG (controls: 0.243 +/- 0.231, p less than 0.001), 0.257 +/- 0.215 g/L for IgA (controls: 0.038 +/- 0.035, p less than 0.001), and 0.282 +/- 0.234 g/L for IgM (controls: 0.191 +/- 0.165, p greater than 0.05). Sensitivity and linearity are such that fivefold dilutions of patients' serum with either a buffered albumin solution or control serum yielded values close to the expected values for IgG. Similarly diluted sera gave inordinately high values in the radiometric binding assay. Neither parametric (linear regression) nor nonparametric correlation methods (Spearman's rank and Kendall's tau) show a significant correlation between patients' data obtained by the present technique and that by a radiometric binding assay (p greater than 0.05), although combined data from patients and controls demonstrate a significant nonparametric correlation (p less than 0.005 for Spearman's and p less than 0.02 for Kendall's).
Discordant inter-kit results in the radioimmunoassay for choriogonadotropin in serum.
Authors: Rzasa P J , Caride V J , Prokop E K
Published: January 2020

Discordant results for concentrations of choriogonadotropin (HCG) in sera from five of 162 women are reported, as determined with four commercial radioassay kits: Becton-Dickinson, Clinetics, Corning, and Hybritech. Results ranged from 11.2 to 36.7 int. units/L, when assayed with the Becton-Dickinson kit, but HCG was not detectable when assayed with the other kits. None of the patients was pregnant, and none had evidence of persistent trophoblastic or nontrophoblastic disease. Concentrations of lutropin, follitropin, thyrotropin, triglycerides, cholesterol, protein, and albumin were normal. Assay of diluted sera did not parallel the standard curve, and nonspecific binding in the patients' samples was negligible. Some kits for HCG may be influenced by inherent methodological factors or interfering serum constituents, which can lead to spurious results.
Multivariate examination of response surfaces around the reaction conditions for the Scandinavian Society's recommended method for creatine kinase determinations.
Authors: Sampson E J , Whitner V S , Ali M , Fast D M
Published: January 2020

We examined the reaction surfaces around five variables (imidazole, ADP, creatine phosphate, magnesium, and pH) in the Scandinavian method for determining creatine kinase, using factorial experimentation (five level, five factor) at reaction temperatures of 30 and 37 degrees C. Theoretical response surfaces were computed by fitting a quadratic polynomial equation to the experimental data by least-squares regression. Essentially no differences were apparent in the theoretical curves among the five specimens we analyzed, or between reaction temperatures. Our response-surface data showed the following: for pH and imidazole, activity optima in the region of the Scandinavian conditions; for creatine phosphate, a broad plateau over the concentration range investigated (10 to 50 mmol/L); and for magnesium and ADP, gently increasing contours with maximal creatine kinase activity at concentrations greater than those investigated in our study (magnesium 15 mmol/L, ADP 3.5 mmol/L).
Purification of lactate dehydrogenase isoenzymes one, two, and three from human erythrocytes.
Authors: Pridgar E M , Moses G C , Henderson A R
Published: January 2020

Lactate dehydrogenase (LD) isoenzymes 1, 2, and 3 were prepared from human erythrocytes by sequential ion-exchange chromatography followed by general-ligand (AMP analog) affinity chromatography. Respective yields, purification factors, and specific activities (kU per gram of protein) were 25%, 4394-fold, and 209.7; 40% 4385-fold, and 199.1; and 18%, 7565-fold, and 192.9. The respective preparations contained less than 0.5% of contaminating LD isoenzyme activity as judged from electrophoresis on thin-layer agarose, were homogeneous as judged by electrophoresis on polyacrylamide gel (both in the presence and absence of sodium lauryl sulfate), and showed minor contamination by other LD isoenzymes as judged by analytical isoelectric focusing. We think that these preparations would be useful as human-based calibrating or reference materials. Their purity is such that these preparations could also be used as antigens for the development of suitable antisera.
Simple screening test for qualitative detection of increased delta-aminolevulinic acid in urine.
Authors: Tishler P V , Winston S H
Published: January 2020

A simple qualitative procedure for demonstrating increased delta-aminolevulinic acid (ALA) in urine is needed to identify individuals with an inherited deficiency of the enzyme ALA dehydratase, a newly described porphyria-like syndrome. The present procedure includes two steps, the last of which is carried out only on samples that are positive in the first step. Urine is tested for pyrroles, preformed or formed by heating with acetylacetone, by looking for a pink color after p-dimethylaminobenzaldehyde is added. Samples showing the color are next tested for ALA by adsorbing natural pyrroles onto Dowex-1 in sodium acetate, heating the supernate with acetylacetone, adsorbing the resulting aminoketone pyrroles onto Dowex-1 in water, separating the ALA from other aminoketones by differential extraction, and again looking for a pink color with p-dimethylaminobenzaldehyde. Positive urines, those with concentrations of ALA greater than or equal to 10-15 mg/L, yield a pink color in each step. Samples with greater than 30 mg/L ALA show a magenta color. Aminoacetone in excess of 3 mg/L ALA also yields a pink color, but such a concentration in urine is unlikely. Many samples can easily be tested concurrently.
Application of enzyme immunoassays to coagulation testing.
Authors: Amiral J , Adalbert B , Adam M
Published: January 2020

Enzyme immunoassays are very useful for the detection of low concentrations of coagulation proteins and pathological markers in plasma. Analytes in the ng/mL range are measurable with good reproducibility with intra- and interassay CVs of less than 5% to 10%. "Sandwich" methods have been developed for von Willebrand factor (plasma concentration about 8 micrograms/mL, Factor IX (5 micrograms/mL), protein C (4 micrograms/mL), and Factor X (10 micrograms/mL). However, this technique is only suitable for macromolecules; for low-molecular-mass peptides such as fibrinopeptide A a competitive method is used. Normal concentrations of fibrinopeptide A are below 3 ng/mL, with greater values suggesting in vivo generation of thrombin; thus this test is quite useful in detecting thrombosis. Reagents for both the sandwich and competitive methods are commercially available and cost effective, and have a longer shelf-life than those for radioimmunoassays.
Comparison of the Nancy Index With Continuous Geboes Score: Histological Remission and Response in Ulcerative Colitis
Authors: Magro Fernando , Lopes Joanne , Borralho Paula , Dias Cláudia Camila , Afonso Joana , Ministro Paula , Santiago Mafalda , Geboes Karel , Carneiro Fátima ,   Portuguese IBD Study Group [GEDII], Lopes Susana , Coelho Rosa , Cotter José , de Castro Francisca Dias , de Sousa Helena Tavares , Salgado Marta , Andrade Patrícia , Vieira Ana , Figueiredo Pedro , Caldeira Paulo , Sousa A , Duarte Maria , Ávila Filipa , Silva João , Moleiro Joana , Mendes Sofia , Giestas Sílvia , Sousa Paula , Gonçalves Raquel , Gonçalves Bruno Moreira , Oliveira Ana Maria , Chagas Cristina
Published: January 2020

Background and Aims

Evidence has been supporting that histological activity of ulcerative colitis [UC] has relevance for the prediction of clinical outcomes in UC patients, such as clinical relapse. In this study, we aimed to compare two histological indexes—the continuous Geboes score [GS] and the Nancy index [NI] —regarding their definitions of histological remission and response, and to determine the ability of faecal calprotectin [FC] levels to discriminate between these histological statuses according to the NI.

Methods

A large cohort of UC patients [N = 422] who were previously enrolled in other studies was analysed.

Results

GS and NI were shown to be strongly correlated [correlation coefficient: 0.882, p <0.001], indicating high accordance in the classification of patients as having/not having histological remission and response. FC levels moderately correlated with NI regarding these histological statuses [correlation coefficient: 0.481, p <0.001], moderately predicted the absence of remission defined by NI >0 {area under the curve (AUC) 0.667 (95% confidence interval [CI] 0.609–0.724)}, and were good predictors of the absence of histological response defined by NI >1 (AUC 0.825 [95% CI 0.777–0.872]). The optimal FC cut-offs determined to predict the NI-defined histological remission and response were 91 μg/g and 106 μg/g, when maximising the negative predictive value [NPV].

Conclusions

Due to the higher applicability of the NI, this study encourages the systematic use of this histological index to assess histological remission and response in UC patients.
OP28 Gene expression (GE) values in a phase 2 trial of mirikizumab in ulcerative colitis (UC) correlate better with histopathology (HP) than endoscopy (EN) and Mayo scores
Authors: Steere B , GOTTLIEB K , Schmitz J , Higgs R , Jia B , Milch C , Tuttle J , Krishnan V , Belin R , Reinisch W
Published: January 2020

Background

HP is considered integral to the definition of mucosal healing of UC. The regulated transcription of genes determines cell identity and function, and, consequentially, HP and EN appearance. Here we correlate GE in a UC clinical trial with EN and HP indices, and the modified Mayo score (MMS).

Methods

249 patients. with moderately-to-severely active UC were randomised to placebo or different doses of mirikizumab, a p19-directed IL-23 antibody (NCT02589665). Gene expression in baseline biopsy samples from the most affected area in the colon was evaluated using Affymetrix HTA2.0 exon-format microarray with a typical Bioconductor workflow (DOI: 10.1186/gb-2004-5-10-r801). Genes were represented by their largest groups of highly correlated exon groups. Expression values were correlated with Robart‘s Histopathology Index (RHI), Geboes score (GS), Ulcerative Colitis Endoscopic Index of Severity (UCEIS), and modified Mayo score (MMS) at baseline. A threshold of false-discovery rate (FDR)-adjusted p-value ≤0.05 was applied to the significance of the r-values. Subsets of genes of interest were subjected to pathway enrichment using MetaCore (Clarivate Analytics). Simulation analysis (SIM) was performed to estimate any bias in correlation estimates caused by the number of levels used for different clinical scores.

Results

The plot (Figure 1) shows more genes with expression values that correlate significantly (q < 0.05) with RHI (4647), than UCEIS (1232), or MMS (869) (refer to legend). There are 498 genes that intersect all scores, representing predominantly the pathways/networks in Table 1. The RHI covers 86 % (1064/1232) of the genes that correlate with UCEIS. The genes unique to the UCEIS (168) do not significantly map to a specific pathway/network. Highest absolute r-values were RHI 0.56, GS 0.45, UCEIS 0.4 and MMS 0.3. The corresponding lowest q values expressed in −Log10 are 15, 9, 4, and 2.5. SIM shows only a minimal bias of <0.03 units (Pearson’s and Spearman’s r).

Conclusion

In our analysis, RHI tracked baseline gene expression values best, followed by another HP index (GS), followed by EN (UCEIS) and finally MMS. Judging by the orders-of-magnitude greater significance of correlation with gene expression values, the histology indices are particularly information-rich. Regulated gene expression, fundamental to a phenotypic appearance in HP, seems to best mirrored by the RHI, followed by GS. The genes that track with all indices predominantly modulate cell adhesion and leukocyte trafficking. The genes that are unique to the UCEIS, compared with the RHI, do not significantly belong to any recognisable pathway or network. Further evaluation of what current EN scores contribute relative to HP disease activity assessment in UC may be useful.
P026 The impact of different dietary factors in the etiopathogenesis of inflammatory bowel disease: a retrospective case–control study
Authors: Preda C M , Manuc T , Chifulescu A E , Istratescu D , Louis E , Diculescu M , Cristian T , Gheorghe L S , Manuc M
Published: January 2020

Background

Inflammatory bowel disease development has been associated with several environmental factors, among which diet can play a key role, probably due to a westernised lifestyle. However, its involvement in the pathogenesis of IBD is difficult to be demonstrated. The aim of this study was to analyse dietary composition in a Romanian and Belgian population with IBD.

Methods

We conducted an observational retrospective comparative study using two European cohorts (Romanian and Belgian). The IBD group included 76 Romanian and 53 Belgian patients with an IBD diagnosis while the control group included 56 healthy persons (35 Romanians and 21 Belgians). All subjects were interviewed and asked to fill in a questionnaire regarding diet.

Results

The baseline characteristics of the patients included in the two cohorts were described in the table. Figures 1 and 2 illustrated the consumption of different foods in Romanian, respectively, Belgian IBD patients before the onset of the disease vs. healthy controls (variables were compared with Fisher’s exact test). In the entire IBD cohort (Romanian+Belgian), we found a significantly increased consumption of sweets (OR 3.36[95% CI 1.6,7]), processed and high-fat meat (OR 2.5[95% CI1.4, 4.7], fried food (OR 9.5[3.8, 23.6]), salt (OR 2.8[1.5, 5.3]), ice cream (OR 3.25[1.1, 9.8]), mayonnaise (OR 3.49[1.1, 10.3]), margarine (OR 5.63[1.64, 19.33]), chips/nachos/other snacks (OR 2.3[0.97, 5.73]), compared with the healthy control group. The intake of seeds, nuts (OR 0.26[0.14,0.52]) and the yoghurt consumption (OR 0.44[0.23, 0.83]) was lower in the IBD group compared with the control group.

Conclusion

In Romanian and Belgian IBD patients, a westernised diet with increased consumption of sweets, processed food, high-fat meat, fried food, salt, margarine, snacks, ice cream and mayonnaise seems to be a risk factor for IBD. Food intake of seeds, nuts and yogurt may be a protective factor.
P035 NUDT15 variance increases DNA-incorporated thiopurine metabolites and lymphocyte apoptosis in patients with inflammatory bowel disease
Authors: Kiyohara H , Toyonaga T , Kuronuma S , Ueno A , Okabayashi S , Ozaki R , Takeuchi O , Coulthard S A , Redfern C P F , Terai H , Tanaka Y , Nakano M , Hibi T , Kobayashi T
Published: January 2020

Background

A novel thiopurine metabolizing enzyme, nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) was associated with drug-induced leukopenia in patients with non-synonymous genetic polymorphisms. Thiopurine-induced leukopenia in Japanese patients with genetic variance in NUDT15 (c.415C>T) appears to be independent of the 6-thioguanine nucleotide concentration in red blood cells. However, detailed molecular mechanism how NUDT15 variance causes thiopurine-induced leukopenia remains unclear and NUDT15-associated subcellular thiopurine metabolism has not been investigated in patients with inflammatory bowel diseases (IBD).

Methods

DNA-incorporated deoxythioguanosine (dTG) was measured in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with IBD under thiopurine treatment. Association of a single-nucleotide polymorphism for NUDT15 (c.415C>T) with dTG in PBMCs (dTGPBMC) was examined. Peripheral blood T lymphocytes were cultured in vitro with 6-thioguanine (6-TG) to examine the Impact of NUDT15 genotypes on incorporation into DNA, cell proliferation and apoptosis.

Results

NUDT15 variants had significantly higher dTGPBMC per thiopurine dosage than non-variants (homozygous variants (TT) vs. heterozygous variants (CT) vs. non-variants (CC), 4418.0 vs. 663.0 vs. 295.3 dTG mol/106 moles dA per mg/kg/day of 6-MP (Figure A)). dTGPBMC and peripheral lymphocyte counts showed a negative correlation (r = −0.30, p = 0.015) (Figure B). Peripheral blood lymphocytes from patients with NUDT15 variance showed a higher DNA-incorporated dTG associated with increased apoptosis (increase of Annexin V+ PI+ CD4+ lymphocytes; TT vs. CT vs. CC, 158.5 % vs. 80.1 % vs. 57.9 % (p = 0.0427)) (Figure C) and decreased proliferation (decrease of proliferative CD4+ lymphocytes, TT vs. CT vs. CC, 49.0 % vs. 25.0 % vs. 19.1 % (p = 0.0098)) (Figure D) when cultured with 6-thioguanine in vitro.

Conclusion

DNA-incorporated dTG affected by NUDT15 genotypes induces T lymphocyte apoptosis in patients with IBD.
P053 3-Oxo-C12:2, a quorum sensing molecule from the human gut, exerts anti-inflammatory effects through a bitter taste receptor
Authors: Coquant G , Aguanno D , Peyrottes A , Brot L , Thenet S , Grill J P , De Sordi L , Seksik P
Published: January 2020

Background

Acyl-homoserine lactones (AHLs) are quorum sensing molecules involved in bacterial communication network and can also have an impact on host’s cells. We recently showed the presence of AHLs in the gut ecosystem and identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in faecal samples of inflammatory bowel disease (IBD) patients, especially during flare, and its absence was correlated to dysbiosis. 3-Oxo-C12:2 is structurally close to an AHL well described and synthesised by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identify which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesised that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor, called T2R138, which is a GPCR expressed by immune and epithelial gut cells.

Methods

To test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-γ (IFN-γ; 20 U/ml) and lipopolysaccharide (LPS; 10 ng/ml). Inflammatory response was monitored by measuring cytokine secretion (TFNα, IL-6 and IL-10) via ELISA. Protein expression was assessed by Western blot. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. Cytotoxicity was checked by measuring LDH release.

Results

In steady state, challenged by 3-oxo-C12:2, RAW264.7 macrophages showed no change in cytokine expression (TNFα, IL-6 and IL-10). After LPS/IFN-γ activation, we observed a decrease in the ratio of secreted TNFα/IL-10 when cells are exposed to 3-oxo-C12:2, in a dose-dependent manner: 15 μM (−30%, p < 0.05), 25 μM (−50%, p < 0.001) and 50 μM (−65%, p < 0.0001). This reflects an anti-inflammatory effect, without increasing cytotoxicity. Those immune-modulatory effects were lost in presence of Probenecid. Moreover, the amount of T2R138 protein was not changed in the presence of Probenecid and/or 3-oxo-C12:2. Therefore, the loss of anti-inflammatory effects with the inhibitor was not due to a decrease of the receptor expression in our experimental conditions.

Conclusion

3-Oxo-C12:2 exerts a dose-dependent anti-inflammatory effect on murine immune cells. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL. We are currently studying via a multiplex assay the cytokine response after exposing cells to 3-oxo-C12:2. Studying the signalling between the receptor and the anti-inflammatory response would allow us to understand better the inter-kingdom dialogue between microbiota and the host and to what extend AHLs are involved.

We are grateful for the FRM for the financial support (ECO201806006843)
P056 Kefir (Lactobacillus kefiranofaciens) promotes autophagy and protects the intestinal barrier against DSS-induced colitis
Authors: Da Silva K N , Cordeiro T S , Fávero A G , Bogsan C S , Sinigaglia R D C , Miszputen S J , Ambrogini–Júnior O , Paiotti A P R
Published: January 2020

Background

Inflammatory bowel disease occurs when luminal antigens from microbiota stimulate the immune system of an intestinal barrier through defects in the tight junctions lead to an exacerbated response in the genetically predisposed individual. It is well known that the consumption of probiotics improves intestinal health. Specifically, the Lactobacillus sp. strains play a key role in the epithelial intestinal barrier, promote mucin secretion, better performance of tight junctions and prevent apoptosis of epithelial cells. This study aimed to evaluate the role of Kefir (Lactobacillus kefiranofaciens) on inflammation of dextran sodium sulphate (DSS)-induced colitis.

Methods

Forty male Wistar rats were randomised into five groups: Sham; 5% DSS-induced colitis; control Kefir; 5% DSS-induced colitis treated with Kefir; 5% DSS-induced colitis treated with Kefir + 5-ASA (125mg/kg/day) (Ethics Committee on the Use of Animals number 3627290119). The lactic culture of Kefir (Danisco Biolacta, Olstyn, Poland) was administered ad libitum per 6 days. The preparation of the matrix was carried out as follows: whole milk powder was reconstituted to 10% with distilled water and was incubated at 85°C for 15 min using a mechanical shaker. Subsequently, 20 mg freeze-dried Kefir culture was added to 100 ml of the treated milk and fermentation were carried out at 23°C for 16 h. When the desired pH of 4.6 was reached, the fermentation was stopped by cooling the flasks in ice bath and storing them under refrigeration at 4°C until utilisation. The rats were euthanised on day 9. Colonic samples were processed for transmission electron microscope (TEM) and conventional microscopy. Samples were analysed in a TEM EX II 1200 (JEOL, Japan) coupled to a digital camera system GATAN Orius (USA). Histopathological evaluation was performed by a pathologist under a light microscope. Parameters such as inflammation extent, regeneration and crypt damage were graded according to Dieleman et al.3 Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Tukey’s test using Graph Pad Prism (version 6.0).

Results

Kefir treatment reduces the severity of DSS-induced colitis evidenced by decreased abdominal discomfort and rectal bleeding. Histologically, there was a significant decreased of colonic damage and preservation of goblet cells (p < 0.0001) compared with DSS-colitis control group (Figure 1). The effect of Kefir was also verified by TEM analysis at the ultrastructural level, demonstrating the tight junctions preserved, decreased of reticulum edema and increased of autophagosomes (Figure 2).

Conclusion

Kefir promotes autophagy and protects the intestinal barrier against damage caused by DSS.
P073 Compartmentalised human gut immunity: studies of innate and adaptive lymphocyte distribution in the epithelium, lamina propria and GALT of healthy gut mucosa by flow cytometry
Authors: Carrasco Garcia A , Rao A , Kokkinou E , Haapaniemi S , Lindforss U , Winberg M E , Casado-Bedmar M , Fenton T , Jørgensen P , Nordenvall C , Agace W , Keita Å V , Söderholm J D , Mjösberg J
Published: January 2020

Background

The human gut mucosal immune system is compartmentalised in distinct and specialised immune niches. The epithelium and the lamina propria have been proposed as effector sites, while gut-associated lymphoid tissues (GALTs) constitute inductive immune niches. The major mucosal GALTs are the Peyer’s patches in the ileum and the colonic isolated lymphoid follicles (ILFs), scattered in the submucosa of the colon. The majority of studies of human gut immune function in health and disease have analysed unfractionated mucosal tissue samples. Hence, in contrast to mice, little is known about compartmentalised immune cell specialisation in the human gut. The aim of this study was to use novel dissection methods to analyse separate human gut immune niches.

Methods

Macroscopically healthy margins from colorectal cancer colectomies were obtained at a minimum distance of 10 cm from the tumour border. After faeces, mucus, fat and muscle removal, Peyer’s patches were identified and dissected using a stereomicroscope (based on Keita et al., Lab Invest, 2006). Colonic mucosa and submucosa (containing ILFs) fractions were mechanically separated by forceps (based on the method developed by Fenton et al., Immunity, under revision). Isolation of epithelial and lamina propria fractions from the mucosal compartment was performed by calcium chelation (DTT and EDTA) and enzymatic digestion (Collagenase II and DNAse), respectively. Cell suspensions from each fraction were analysed by flow cytometry (BD LSR-Fortessa and BD FACSymphony).

Results

As expected, mucosal GALTs were characterised by an enrichment of germinal centre B cells (CD19+CD20+CD38+), lymphoid tissue-like innate lymphoid cells (Lin−CD127+CD117+Nrp1+) and a higher CD4+/CD8+ T-cell ratio vs. mucosa, whereas the mucosal fraction was enriched for plasma cells (CD19+CD20−CD38high) and distinguished by a decreased CD4+/CD8+ T-cell ratio as compared with the GALT in both ileum and colon. CD19+/CD3+ ratios were only higher in Peyer’s patches but not in colonic submucosa enriched with ILFs, possibly due to the smaller size of the B-cell follicles in the latter. The intraepithelial compartment lacked B cells and contained more γδ-T cells as compared with the GALT and lamina propria.

Conclusion

We have used novel dissection methods in human intestinal tissues that reveal a compartmentalised immune cell specialisation that is in line with what has previously been described in mice. The method will allow for future deeper analysis of the human gut immune niches in health and disease, such as in inflammatory bowel disease.
P181 Triage straight to test (tSTT) improves early diagnosis of inflammatory bowel disease (IBD)
Authors: Jora S
Published: January 2020

Background

It is well known that there is a significant delay in diagnosing IBD. On average it takes 4–9 months to diagnose Crohn’s disease (CD) and 4 months for ulcerative colitis (UC)1. Barts Health NHS Trust in the UK is one of the pioneer hospitals in establishing Triage Straight to Test (tSTT) for patients with lower gastrointestinal (GI) symptoms. The aim of the programme is to reduce the time from a primary care physician (General practitioner) referral to the secondary care to achieve a definitive diagnosis. This minimises the number of outpatient appointments required and makes an efficient use of medical and surgical resources. We aim to study whether tSTT pathway reduces the time to diagnose IBD.

Methods

Patients with lower GI symptoms were referred to a specialist tSTT triage nurse, where they were allocated into two pathways—routine (18-week wait) and upgraded (2-week wait). A comprehensive telephone assessment, calprotectin levels and clinical information provided by the GP would determine the route and urgency of the investigation. Pathologies such as cancer, IBD and polyps were treated via a predefined management pathway. The patients with a benign disease were discharged and in some cases called for a follow-up appointment.

Results

During July 2013 to November 2019 a total of 2270 patients have been triaged. One hundred and one (4.45%) patients were diagnosed with IBD, where 36 patients (36%) were histologically characterised as CD and the other 65 patients (64%) were diagnosed as UC. The mean age of diagnosis for both CD and UC patients was 40 years (range 16–79). The average time from referral to endoscopy on tSTT pathway was 22.6 days for upgraded referrals and 43.5 days for non-upgraded. There is a significant difference in time to diagnosis between routine and upgraded pathways (22.6 days vs. 43.5 days, p < 0.001).

Conclusion

The above data suggest a significant reduction in time to diagnose IBD through tSTT pathway. Early diagnosis ensures that the patient receives an early customised care and avoids disease-related complications. It also means that the NHS resources can be used wisely by avoiding a delay in getting outpatient consultant appointments.